(19)
Europaisches Patentamt
European Patent Office
Office europeen des brevets
(11)
EP0 774 511 A1
(12)
EUROPEAN PATENT APPLICATION
(43) Date of publication:
21.05.1997 Bulletin 1997/21
(21) Application number: 96112510.1
(22) Date of filing: 10.07.1991
(51) mtci e : C12N 15/10, C12N 15/62,
C12N 15/73, C07K 16/00,
O I zll\J I Id. I
/ / A r\ K 1 A lf~\ A A /"> n A . A f~\\
II (C12N1/21 , C12R1 :19)
(84) Designated Contracting States:
• Griffiths, Andrew David
AT BE CH DE DK ES FR GB GR IT LI LU NL SE
Cambridge, CB1 4AY (GB)
• Jackson, Ronald Henry
(30) Priority: 10.07.1990 GB 9015198
Cambridge, CB1 2NU (GB)
19.10.1990 GB 9022845
• Holliger, Kaspar Philipp
12.11.1990 GB 9024503
Cambridge, CB1 4HT (GB)
06.03.1991 GB 9104744
• Marks, James David
15.05.1991 GB 91 10549
Kensington, CA 94707-1310 (US)
• Clackson, Timothy Piers
(62) Application number of earlier application in
somerville, MA 02143 (US)
accordance with Art. 76 EPC: 91913039.3
• Chiswell, David John
Middle Claydon, Buckingham, MK18 2LD (GB)
(71) Applicants:
• Winter, Gregory Paul
• CAMBRIDGE ANTIBODY TECHNOLOGY
Cambridge, CB2 1TQ (GB)
LIMITED
• Bonnert, Timothy Peter
Melbourn, Cambridgeshire SG8 6EJ (GB)
Seattle, WA 981 02 (US)
• MEDICAL RESEARCH COUNCIL
London W1N4AL (GB)
(74) Representative: Walton, Sean Malcolm et al
Mewburn Ellis
111 V ¥¥ *>mT Wi III ^mm III *m** m
(72) Inventors:
York House,
• McCafferty, John
23 Kingsway
Babraham, CB2 4AP (GB)
London WC2B 6HP (GB)
• Pope, Anthony Richard
Cambridge, CB1 2LW(GB)
Remarks:
• Johnson, Kevin Stuart
This application was filed on 02 - 08 - 1 996 as a
Caldecote, Highf ields, Cambridge CB3 7NY (GB)
divisional application to the application mentioned
• Hoogenboom, Hendricus Renerus Jacobus
under INID code 62.
Mattheus
6214 AE Maastricht (NL)
(54) Methods of producing members of specific binding pairs
LO
CL
LU
(57) A member of a specific binding pair (sbp) is
identified by expressing DNA encoding a genetically di-
verse population of such sbp members in recombinant
host cells in which the sbp members are displayed in
functional form at the surface of a secreted recombinant
genetic display package (rgdp) containing DNA encod-
ing the sbp member or a polypeptide component there-
of, by virtue of the sbp member or a polypeptide com-
ponent thereof being expressed as a fusion with a cap-
sid component of the rgdp. The displayed sbps may be
selected by affinity with a complementary sbp member,
and the DNA recovered from selected rgdps for expres-
sion of the selected sbp members. Antibody sbp mem-
bers may be thus obtained, with the different chains
thereof expressed, one fused to the capsid component
and the other in free form for association with the fusion
partner polypeptide. A phagemid may be used as an ex-
pression vector, with said capsid fusion helping to pack-
age the phagemid DNA. Using this method libraries of
DNA encoding respective chains of such multimeric sbp
members may be combined, thereby obtaining a much
greater genetic diversity in the sbp members than could
easily be obtained by conventional methods.
Printed by Jouve, 75001 PARIS (FR)
(Cont. next page)
EP0 774 511 A1
Fig.l
dAb H Fab
2
EP0 774 511 A1
Description
The present invention relates to methods for producing members of specific binding pairs. The present invention
also relates to the biological binding molecules produced by these methods.
5 Owing to their high specificity for a given antigen, the advent of monoclonal antibodies (Kohler, G. and Milstein C;
1975 Nature 256: 495) represented a significant technical break-through with important consequences both scientifi-
cally and commercially.
Monoclonal antibodies are traditionally made by establishing an immortal mammalian cell line which is derived
from a single immunoglobulin producing cell secreting one form of a biologically functional antibody molecule with a
10 particular specificity. Because the antibody-secreting mammalian cell line is immortal, the characteristics of the antibody
are reproducible from batch to batch. The key properties of monoclonal antibodies are their specificity for a particular
antigen and the reproducibility with which they can be manufactured.
Structurally, the simplest antibody (IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L)
chains inter-connected by disulphide bonds (see figure 1 ). The light chains exist in two distinct forms called kappa (K)
15 and lambda (X). Each chain has a constant region (C) and a variable region (V). Each chain is organized into a series
of domains. The light chains have two domains, corresponding to the C region and the other to the V region. The heavy
chains have four domains, one corresponding to the V region and three domains (1,2 and 3) in the C region. The
antibody has two arms (each arm being a Fab region), each of which has a VL and a VH region associated with each
other. It is this pair of V regions (VL and VH) that differ from one antibody to another (owing to amino acid sequence
20 variations), and which together are responsible for recognising the antigen and providing an antigen binding site (ABS).
In even more detail, each V region is made up from three complementarity determining regions (CDR) separated by
four framework regions (FR). The CDR's are the most variable part of the variable regions, and they perform the critical
antigen binding function. The CDR regions are derived from many potential germ line sequences via a complex process
involving recombination, mutation and selection.
25 it has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example
binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CHI domains; (ii) the Fd fragment consisting
of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody,
(iv) the dAb fragment (Ward, E.S. etal., Nature 341, 544-546(1989) which consists of a VH domain: (v) isolated CDR
regions; and (vi) F(ab') 2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at
30 the hinge region.
Although the two domains of the Fv fragment are coded for by separate genes, it has proved possible to make a
synthetic linker that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bird, R.E.
et al., Science 242, 423-426 (1988) Huston, J.S. at al., Proc. Natl. Acad. Sci., USA 85, 5879-5883 (1988)) by recom-
binant methods. These scFv fragments were assembled from genes from monoclonals that had been previously iso-
35 lated. In this application, the applicants describe a process to assemble scFv fragments from VH and VL domains that
are not part of an antibody that has been previously isolated.
Whilst monoclonal antibodies, their fragments and derivatives have been enormously advantageous, there are
nevertheless a number of limitations associated with them.
Firstly, the therapeutic applications of monoclonal antibodies produced by human immortal cell lines holds great
40 promise for the treatment of a wide range of diseases (Clinical Applications of Monoclonal Antibodies. Edited by E. S.
Lennox. British Medical Bulletin 1984. Publishers Churchill Livingstone). Unfortunately, immortal antibody-producing
human cell lines are very difficult to establish and they give low yields of antibody (approximately 1 |~ig/ml). In contrast,
equivalent rodent cell lines yield high amounts of antibody (approximately 100 |ug/ml). However, the repeated admin-
istration of these foreign rodent proteins to humans can lead to harmful hypersensitivity reactions. In the main therefore,
45 these rodent-derived monoclonal antibodies have limited therapeutic use.
Secondly, a key aspect in the isolation of monocional antibodies is how many different clones of antibody producing
cells with different specificities, can be practically established and sampled compared to how many theoretically need
to be sampled in order to isolate a cell producing antibody with the desired specificity characteristics (Milstein, C,
Royal Soc. Croonian Lecture, Proc. R. Soc. London, B. 239; 1-16, (1990)). For example, the number of different spe-
50 cificities expressed at any one time by lymphocytes of the murine immune system is thought to be approximately 10 7
and this is only a small proportion or the potential repertoire of specificities. However, during the isolation of a typical
antibody producing cell with a desired specificity, the investigator is only able to sample 1 0 3 to 1 0 4 individual specificities.
The problem is worse in the human, where one has approximately 10 12 lymphocyte specificities, with the limitation on
sampling of 10 3 or 10 4 remaining.
55 This problem has been alleviated to some extent in laboratory animals by the use of immunisation regimes. Thus,
where one wants to produce monoclonal antibodies having a specificity against a particular epitope, an animal is
immunised with an immunogen expressing that epitope. The animal will then mount an immune response against the
immunogen and there will be a proliferation of lymphocytes which have specificity against the epitope. Owing to this
3
EP0 774 511 A1
proliferation of lymphocytes with the desired specificity, it becomes easier to detect them in the sampling procedure.
However, this approach is not successful in all cases, as a suitable immunogen may not be available. Furthermore,
where one wants to produce human monoclonal antibodies (eg for therapeutic administration as previously discussed),
such an approach is not practically, or ethically, feasible.
5 In the last few years, these problems have in part, been addressed by the application of recombinant DNA methods
to the isolation and production of e.g. antibodies and fragments of antibodies with antigen binding ability, in bacteria
such as E.coli .
This simple substitution of immortalised cells with bacterial cells as the 'factory', considerably simplifies procedures
for preparing large amounts of binding molecules. Furthermore, a recombinant production system allows scope for
10 producing tailor-made antibodies and fragments thereof. For example, it is possible to produce chimaeric molecules
with new combinations of binding and effector functions, humanised antibodies (e.g. murine variable regions combined
with human constant domains or murine-antibody CDRs grafted onto a human FR) and novel antigen-binding mole-
cules. Furthermore, the use of polymerase chain reaction (PCR) amplification (Saiki, R.K., at al., Science 239 , 487-491
(1 988)) to isolate antibody producing sequences from cells (e.g. hybridomas and B cells) has great potential for speed-
's ing up the timescale under which specificities can be isolated. Amplified VH and VL genes are cloned directly into
vectors for expression in bacteria cr mammalian cells (Orlandi, R., at al., 1989, Proc. Natl. Acad. Sci., USA 86,
3833-3837; Ward, E.S., et al., 1989 supra; Larrick, J.W., et al., 1989, Biochem. Biophys. Res. Commun. 160,
1250-1255; Sastry, L. at al., 1989, Proc. Natl. Acad. Sci., USA., 86, 5728-5732). Soluble antibody fragments secreted
from bacteria are then screened for binding activities.
20 However, like the production system based upon immortalised cells, the recombinant production system still suffers
from the selection problems previously discussed and therefore relies on animal immunization to increase the propor-
tion of cells with desired specificity. Furthermore, some of these techniques can exacerbate the screening problems.
For example, large separate H and L chain libraries have been produced from immunized mice and combined together
in a random combinatorial manner prior to screening (Huse, W.D. et al., 1989, Science 246 , 1275-1281 , WO90/14443;
25 W090/14424 and WO90/14430). Crucially however, the information held within each cell, namely the original pairing
of one L chain with one H chain, is lost. This loses some, of the advantage gained by using immunization protocols in
the animal. Currently, only libraries derived from single VH domains (dAbs; Ward, E.S., et al., 1989, supra.) do not
suffer this drawback. However, because not all antibody VH domains are capable of binding antigen, more have to be
screened. In addition, the problem of directly screening many different specificities in prokaryotes remains to be solved.
30 Thus, there is a need for a screening system which ameliorates or overcomes one or more of the above or other
problems. The ideal system would allow the sampling of very large numbers of specificities (eg 1 0 6 and higher); rapid
sorting at each cloning round, and rapid transfer of the genetic material coding for the binding molecule from one stage
of the production process, to the next stage.
The most attractive candidates for this type of screening, would be prokaryotic organisms (because they grow
35 quickly, are relatively simple to manipulate and because large numbers of clones can be created) which express and
display at their surface a functional binding domain eg. an antibody, receptor, enzyme etc. In the UK patent GB
21 37631 B methods for the co-expression in a single host cell of the variable H and L chain genes of immunoglobulins
were disclosed. However, the protein was expressed intracellular^ and was insoluble. Further, the protein required
extensive processing to generate antibody fragments with binding activity and this generated material with only a
40 fraction of the binding activity expected for antibody fragments at this concentration. It has already been shown that
antibody fragments can be secreted through bacterial membranes with the appropriate signal peptide (Skerra, A. and
Pluckthun, A. 1 988 Science 240 1 038-1 040; Better, M at al 1 988, Science 240 1 041 -1 043) with a consequent increase
in the binding activity of antibody fragments. These methods require screening of individual clones for binding activity
in the same way as do mouse monoclonal antibodies.
45 it has not been shown however, how a functional binding domain eg an antibody, antibody fragment, receptor,
enzyme etc can be held on the bacterial surface in a configuration which allows sampling of say its antigen binding
properties and selection for clones with desirable properties. In large part, this is because the bacterial surface is a
complex structure, and in the gram-negative organisms there is an outer wall which further complicates the position.
Further, it has not been shown that eg an antibody domain will fold correctly when expressed as a fusion with a surface
50 protein of bacteria or bacteriophage.
Bacteriophage are attractive prokaryote related organisms for this type of screening. In general, their surface is a
relatively simple structure, they can be grown easily in large numbers, they are amenable to the practical handling
involved in many potential mass screening programmes, and they carry genetic information for their own synthesis
within a small, simple package. The difficulty has been to practically solve the problem of how to use bacteriophages
55 in this manner. A Genex Corporation patent application number WO88/06630 has proposed that the bacteriophage
lambda would be a suitable vehicle for the expression of antibody molecules, but they do not provide a teaching which
enables the general idea to be carried out. For example WO88/06630 does not demonstrate that any sequences: (a)
have been expressed as a fusion with gene V; (b) have been expressed on the surface of lambda; and (c) have been
4
EP0 774 511 A1
expressed so that the protein retains biological activity. Furthermore there is no teaching on how to screen for suitable
fusions. Also, since the lambda virions are assembled within the cell, the fusion protein would be expressed intracel-
lular^ and would be predicted to be inactive. Bass et al., in December 1990 (after the earliest priority date for the
present application) describe deleting part of gene III of the filamentous bacteriophage M13 and inserting the coding
5 sequence for human growth hormone (hGH) into the N-terminal site of the gene. The growth hormone displayed by
M13 was shown to be functional. (Bass, S., at al. Proteins, Structure, Function and Genetics (1990) 8: 309-314). A
functional copy of gene III was always present in addition, when this fusion was expressed. A Protein Engineering
Corporation patent application WO90/02809 proposes the insertion of the coding sequence for bovine pancreatic
trypsin inhibitor (BPTI) into gene VIII of M1 3. However, the proposal was not shown to be operative. For example, there
10 is no demonstration of the expression of BPTI sequences as fusions with protein VIII and display on the surface of
M13. Furthermore this document teaches that when a fusion is made with gene III, it is necessary to use a second
synthetic copy of gene III, so that some unaltered gene III protein will be present. The embodiments of the present
application do not do this. In embodiments where phagemid is rescued with M13K07 gene III deletion phage, there is
no unaltered gene III present.
15 WO90/02809 also teaches that phagemids that do not contain the full genome of M13 and require rescue by
coinfection with helper phage are not suitable for these purposes because coinfection could lead to recombination.
In all embodiments where the present applicants have used phagemids, they have used a helper phage and the
only sequences derived from filamentous bacteriophage in the phagemids are the origin of replication and gene III
sequences.
20 W090/02809 also teaches that their process needed information such as nucleotide sequence of the starting mol-
ecule and its three-dimensioned structure. The use of a pre-existing repertoire of binding molecules to select for a
binding member, such as is disclosed herein, for example using an immunoglobulin gene repertoire of animals, was
not disclosed. Further, they do not discuss favouring variegation of their binding molecules in natural blocks or variation
such as CDRs of immunoglobulins, in order to favour generation of improved molecules and prevent unfavourable
25 variations. WO90/02809 also specifically excluded the application of their process to the production of scFv molecules.
In each of the above discussed patents (WO88/06630 and WO90/02809), the protein proposed for display is a
single polypeptide chain. There is no disclosure of a method for the display of a dimeric molecule by expression of one
monomer as a fusion with a capsid protein and the other protein in a free form.
Another disclosure published in May 1 991 (after the earliest priority date for the present application) describes the
30 insertion into gene VIII of M1 3, the coding sequences for one of the two chains of the Fab portion of an antibody with
co-expression of the other from a plasmid. The two chains were demonstrated as being expressed as a functional Fab
fragment on the surface of the phage (Kang A.S. at al., (1 991 ) Proc. Natl. Acad. Sci, USA, 88 p4363-4366). No dis-
closure was made of the site of insertion into gene VIII and the assay for pAb binding activity by ELISA used a reagent
specific for antibody L chain rather than for phage. A further disclosure published in March 1991 (after the earliest
35 priority date for the present application) describes the insertion of a fragment of the AIDS virus protein gag into the N-
terminal portion of gene III of the bacteriophage fd. The expression of the gag protein fragment was detected by im-
munological methods, but it was not shown whether or not the protein was expressed in a functional form (Tsunetsugu-
Yokota Y at al. (1 991 ) Gene 99 p261 -265).
The problem of how to use bacteriophages in this way is in fact a difficult one. The protein must be inserted into
40 the phage in such a way that the integrity of the phage coat is not undermined, and the protein itself should be functional
retaining its biological activity with respect to antigen binding. Thus, where the protein of choice is an antibody it should
fold efficiently and correctly and be presented for antigen binding. Solving the problem for antibody molecules and
fragments would also provide a general method for any biomolecule which is a member of a specific binding pair e.g.
receptor molecules and enzymes.
45 Surprisingly, the applicants have been able to construct a.bacteriophage that expresses and displays at its surface
a large biologically functional binding molecule (eg antibody fragments, and enzymes and receptors) and which remains
intact and infectious. The applicants have called the structure which comprises a virus particle and a binding molecule
displayed at the viral surface a 'package 1 . Where the binding molecule is an antibody, an antibody derivative or fragment,
or a domain that is homologous to an immunoglobulin domain, the applicants call the package a 'phage antibody' (pAb).
50 However, except where the context demands otherwise, where the term phage antibody is used generally, it should
also be interpreted as referring to any package comprising a virus particle and a biologically functional binding molecule
displayed at the viral surface.
pAbs have a range of applications in selecting antibody genes encoding antigen binding activities. For example,
pAbs could be used for the cloning and rescue of hybridomas (Orlandi, R., et al (1989) PNAS 86 p3833-3837), and in
55 the screening of large combinatorial libraries (such as found in Huse, W.D. et al., 1989, Science 246 , 1275-1281). In
particular, rounds of selection using pAbs may help in rescuing the higher affinity antibodies from the latter libraries. It
may be preferable to screen small libraries derived from antigen-selected cells (Casali, P., et al., (1986) Science 234
p476-479) to rescue the original VH/VL pairs comprising the Fv region of an antibody. The use of pAbs may also allow
5
EP0 774 511 A1
the construction of entirely synthetic antibodies. Furthermore, antibodies may be made which have some synthetic
sequences e.g. CDRs, and some naturally derived sequences. For example, V-gene repertoires could be made in vitro
by combining un-rearranged V genes, with D and J segments. Libraries of pAbs could then be selected by binding to
antigen, hvpermutated in vitro in the antigen-binding loops or V domain framework regions, and subjected to further
5 rounds of selection and mutagenesis.
As previously discussed, separate H and L chain libraries lose the original pairing between the chains. It is difficult
to make and screen a large enough library for a particularly advantageous combination of H and L chains.
For example, in a mouse there are approximately 10 7 possible H chains and 10 7 possible L chains. Therefore,
there are 10 14 possible combinations of H and L chains, and to test for anything like this number of combinations one
10 would have to create and screen a library of about 10 14 clones. This has not previously been a practical possibility.
The present invention provides a number of approaches which ameliorate this problem.
In a first approach, (a random combinatorial approach, see examples 20 and 21 ) as large a library as is practically
possible is created which expresses as many of the 1 0 14 potential combinations as possible. However, by virtue of the
expression of the H and L chains on the surface of the phage, it is reasonably practicable to select the desired com-
15 bination, from all the generated combinations by affinity techniques (see later for description of selection formats).
In a second approach (called a dual combinatorial approach by the present applicants, see example 26), a large
library is created from two smaller libraries for selection of the desired combination. This ameliorates the problems still
further. The approach involves the creation of: (i) a first library of say 10 7 e.g. H chains which are displayed on a
bacteriophage (as a fusion with the protein encoded by gene III) which is resistant to e.g. tetracycline; and (ii) a second
20 Librarv of say 10 7 e.g. L chains in which the coding sequences for these light chains are within a plasmid vector
containing an origin of replication for a bacteriophaae (a phagemid) which is resistant to e.g. ampicillin (i.e. a different
antibiotic) and are expressed in the periplasmic space of a host bacterium. The first library is then used to infect the
bacteria containing the second library to provide 10 14 combinations of H and L chains on the surface of the resulting
phage in the bacterial supernatant.
25 The advantage of this approach is that two separate libraries of eg 10 7 are created in order to produce 10 14 com-
binations. Creating a 10 7 library is a practical possibility.
The 1 0 14 combinations are then subjected to selection (see later for description of selection formats) as disclosed
by the present application. This selection will then produce a population of phages displaying a particular combination
of H and L chains having the desired specificity. The phages selected however, will only contain DNA encoding one
30 partner of the paired H and L chains (deriving from either the phage or phagemid). The sample eluate containing the
population is then divided into two portions. A first portion is grown on e.g. tetracycline plates to select those bacteri-
ophage containing DNA encoding H chains which are involved in the desired antigen binding. A second portion is
grown on e.g. ampicillin plates to select those bacteriophage containing phagemid DNA encoding L chains which are
involved in the desired antigen binding. A set of colonies from individually isolated clones e.g. from the tetracycline
35 plates are then used to infect specific colonies e.g. from the ampicillin plates. This results in bacteriophage expressing
specific combinations of H and L chains which can then be assayed for antigen binding.
In a third approach (called a hierarchical dual combinational approach by the present applicants), an individual
colony from either the H or L chain clone selected by growth on the antibiotic plates, is used to infect a complete library
of clones encoding the other chain (H or L). Selection is as described above. This favours isolation of the most favour-
40 able combination.
In a fourth approach (called a hierarchrical approach by the present applicants, see examples 22 and 46) both
chains are cloned into the same vector. However, one of the chains which is already known to have desirable properties
is kept fixed. A library of the complementary chain is inserted into the same vector. Suitable partners for the fixed chain
are selected following display on the surface of bacteriophage.
45 in a fifth approach (see example 48), to improve the chances of recovering original pairs, the complexity of the
combinatorial libraries can be reduced by using small B populations of B-lymphocytes selected for binding to a desired
antigen. The cells provide e.g. mRNA or DNA, for preparing libraries of antibody genes for display on phage. This
technique can be used in combination with the above mentioned four approaches for selection of antibody specificities.
Phagemids have been mentioned above. The applicants have realised and demonstrated that in many cases
50 phagemids will be preferred to phage for cloning antibodies because it is easier to use them to generate more com-
prehensive libraries of the immune repertoire. This is because the phagemid DNA is approximately 100 times more
efficient than bacteriophage DNA in transforming bacteria (see example 19). Also, the use of phagemids gives the
ability to vary the number of gene III bindinq moecule fusion proteins displayed on the surface of the bacteriophage
(see example 17). For example, in a system comprising a bacterial cell containing a phagemid encoding a gene III
55 fusion protein and infected with a helper phage, induction of expression of the gene 1 1 1 fusion protein to different extents,
will determine the number of gene III fusion proteins present in the space defined between the inner and outer bacterial
membranes following superinfection. This will determine the ratio of gene III fusion protein to native gene III protein
displayed by the assembled phage.
6
EP0 774 511 A1
Expressing a single fusion-protein per virion may aid selection of antibody specificities on the basis of affinity by
avoiding the 'avidity' effect where a phage expressing two copies of a low affin ity antibody would have the same apparent
affinity as a phage expressing one copy of a higher affinity antibody. In some cases however, it will be important to
display all the gene III molecules derived by superinfection of cells containing phagemids to have fusions (e.g. for
5 selecting low affinity binding molecules or improving sensitivity on ELISA). One way to do this is to superinfect with a
bacteriophage which contains a defective gene III. The applicants have therefore developed and used a phage which
is deleted in gene III. This is completely novel.
The demonstration that a functional antigen-binding domain can be displayed on the surface of phage, has impli-
cations beyond the construction of novel antibodies. For example, if other protein domains can be displayed at the
10 surface of a phage, phage vectors could be used to clone and select genes by the binding properties of the displayed
protein. Furthermore, variants of proteins, including epitope libraries built into the surface of the protein, could be made
and readily selected for binding activities. In effect, other protein architectures might serve as "nouvelle" antibodies.
The technique provides the possibility of building antibodies from first principles, taking advantage of the structural
framework on which the antigen binding loops fold. In general, these loops have a limited number of conformations
15 which generate a variety of binding sites by alternative loop combinations and by diverse side chains. Recent successes
in modelling antigen binding sites augurs well for de novo design. In any case, a high resolution structure of the antigen
is needed. However, the approach is attractive for making e.g. catalytic antibodies, particularly for small substrates.
Here side chains or binding sites for prosthetic groups might be introduced, not only to bind selectively to the transition
state of the substrate, out also to participate directly in bond making and breaking. The only question is whether the
20 antibody architecture, specialised for binding, is the best starting point for building catalysts. Genuine enzyme archi-
tectures, such as the triose phosphate isomerase (TIM) barrel, might be more suitable. Like antibodies, TIM enzymes
also have a framework structure (a barrel of p-strands and a-helices) and loops to bind substrate. Many enzymes with
a diversity of catalytic properties are based on this architecture and the loops might be manipulated independently on
the frameworks for design of new catalytic and binding properties. The phage selection system as provided by the
25 present disclosure can be used to select for antigen binding activities and the CDR loops thus selected, used on either
an antibody framework or a TIM barrel framework. Loops placed on a e.g. a TIM barrel framework could be further
modified by mutagenesis and subjected to further selection. Thus, there is no need to select for high affinity binding
activities in a single step. The strategy of the immune system, in which low affinity evolves to high affinity seems more
realistic and can be mimicked using this invention.
30 One class of molecules that could be useful in this type of application are receptors. For example, a specific receptor
could be displayed on the surface of the phage such that it would bind its ligand. The receptor could then be modified
by, for example, jn vitro mutagenesis and variants having higher binding affinity for the ligand selected. The selection
may be carried out according to one or more of the formats described below with reference to figure 2 (which refers
particularly to pAbs) in which the pAb antibody is replaced with a phage receptor and the antigen with a ligand 1 .
35 Alternatively, the phage-receptor could be used as the basis of a rapid screening system for the binding of ligands,
altered ligands, or potential drug candidates. The advantages of this system namely of simple cloning, convenient
expression, standard reagents and easy handling makes the drug screening application particularly attractive. In the
context of this discussion, receptor means a molecule that binds a specific, or group of specific, ligand(s). The natural
receptor could be expressed on the surface of a population of cells, or it could be the extracellular domain of such a
40 molecule (whether such a form exists naturally or not), or a soluble molecule performing a natural binding function in
the plasma, or within a cell or organ.
Another possibility, is the display of an enzyme molecule or active site of an enzyme molecule on the surface of
a phage (see examples 11,12,30,31,32 and 36). Once the phage enzyme is expressed, it can be selected by affinity
chromatography, for instance on columns derivatized with transition state analogues. If an enzyme with a different or
45 modified specificity is desired, it may be possible to mutate an enzyme displayed as a fusion on bacteriophage and
then select on a column derivatised with an analogue selected to have a higher affinity for an enzyme with the desired
modified specificity.
Although throughout this application, the applicants discuss the possibility of screening for higher affinity variants
of pAbs, they recognise that in some applications, for example low affinity chromatography (Ohlson, S. at al Anal.
50 Biochem. 169, p204-208 (1988)), it may be desirable to isolate lower affinity variants.
Examples 21 and 23 show that the present invention provides a way of producing antibodies with low affinities (as
seen in the primary immune response or in unimmunised animals). This is made possible by displaying multiple copies
of the antibody on the phage surface in association with gene III protein. Thus, pAbs allow genes for these antibodies
to be isolated and if necessary, mutated to provide improved antibodies.
55 pAbs also allow the selection of antibodies for improved stability. It has been noted for many antibodies, that yield
and stability are improved when the antibodies are expressed at 30°C rather than 37°C. If pAbs are displayed at 37°C,
only those which are stable will be available for affinity selection. When antibodies are to be used in vivo for therapeutic
or diagnostic purposes, increased stability would extend the half-life of antibodies in circulation.
7
EP0 774 511 A1
Although stability is important for all antibodies and antibody domains selected using phage, it is particularly im-
portant for the selection of Fv fragments which are formed by the non-covalent association of VH and VL fragments.
Fv fragments have a tendency to dissociate and have a much reduced half -life in circulation compared to whole anti-
bodies. Fv fragments are displayed on the surface of phage, by the association of one chain expressed as a gene III
5 protein fusion with the complementary chain expressed as a soluble fragment. If pairs of chains have a high tendency
to dissociate, they will be much less likely to be selected as pAbs. Therefore, the population will be enriched for pairs
which do associate stably. Although dissociation is less of a problem with Fab fragments, selection would also occur
for Fab fragments which associate stably. pAbs allow selection for stability to protease attack, only those pAbs that
are not cleaved by proteases will be capable of binding their ligand and therefore populations of phage will be enriched
10 for those displaying stable antibody domains.
The technique of displaying binding molecules on the phage surface can also be used as a primary cloning system.
For example, a cDNA library can be constructed and inserted into the bacteriophage and this phage library screened
for the ability to bind a ligand. The ligand/binding molecule combination could include any pair of molecules with an
ability to specifically bind to one another e.g. receptor/ligand, enzyme/substrate (or analogue), nucleic acid binding
15 protein/nucleic acid etc. If one member of the complementary pair is available, this may be a preferred way of isolating
a clone for the other member of the pair.
It will often be necessary to increase the diversity of a population of genes cloned for the display of their proteins
on phage or to mutate an individual nucleotide sequence. Although jn vitro or jn vivo mutagenesis techniques could
be used for either purpose, a particularly suitable method would be to use mutator strains. A mutator strain is a strain
20 which contains a genetic defect which causes DNA replicated within it to be mutated with respect to its parent DNA.
Hence if a population of genes as gene III fusions is introduced into these strains it will be further diversified and can
then be transferred to a non-mutator strain, if desired, for display and selection. Example 38 covers the use of mutator
strains with phage antibodies (an example of in vitro mutagenesis and selection of phage antibodies is given in example
45).
25
Targeted gene transfer
A useful and novel set of applications makes use of the binding protein on the phage to target the phage genome
to a particular cell or group of cells. For example, a pAb specific for a cell surface molecule could be used to bind to
30 the target cell via the surface molecule. The phage could then be internalised, either through the action of the receptor
itself or as the result of another event (e.g. an electrical discharge such as in the technique of electroporation). The
phage genome would then be expressed if the relevant control signals (for transcription and translation and possibly
replication) were present. This would be particularly useful if the phage genome contained a sequence whose expres-
sion was desired in the target cell (along with the appropriate expression control sequences). A useful sequence might
35 confer antibiotic resistance to the recipient cell or label the cell by the expression of its product (e.g. if the sequence
expressed a detectable gene product such as a luciferase, see White, M, et al, Techniques 2(4), p1 94-201 (1 990)), or
confer a particular property on the target cell (e.g. if the target cell was a tumour cell and the new sequence directed
the expression of a tumour suppressing gene), or express an antisense construct designed to turn off a gene or set of
genes in the target cell, or a gene or gene product designed to be toxic to the target cell.
40 Alternatively, the sequence whose expression is desired in the target cell can be encoded on a phagemid. The
phagemid DNA may then be incorporated into a phage displaying an antibody specific for a cell surface receptor. For
example, incorporation may be by superinfection of bacteria containing the phagemid, with a helper phage whose
genome encodes the antibody fragment specific for the target cell. The package is then used to direct the phagemid
to the target cell.
45 This technique of "targeted gene transfer" has a number of uses in research and also in therapy and diagnostics.
For example, gene therapy often aims to target the replacement gene to a specific cell type that is deficient in its activity.
Targetting pAbs provide a means of achieving this.
In diagnostics, phage specific for particular bacteria or groups of bacteria have been used to target marker genes,
e.g. luciferase, to the bacterial host (sec, for example, Ulitzer, S., and Kuhn, J., EPA 8530391 3.9). If the host range of
50 the phage is appropriate, only those bacteria that are being tested for, will be infected by the phage, express the
luciferase gene and be detected by the light they emit. This system has been used to detect the presence of Salmonella.
One major problem with this approach is the initial isolation of a bacteriophage with the correct host range and then
the cloning of a luciferase gene cassette into that phage, such that it is functional. The pAb system allows the luciferase
cassette to be cloned into a well characterised system (filamentous phage) and allows simple selection of an appropriate
55 host range, by modifying the antibody (or other binding molecule) specificity that the pAb encodes.
The present applicants have also been able to develop novel selection systems and assay formats which depend
on the unique properties of these replicable genetic display packages e.g. pAbs.
8
EP0 774 511 A1
TERMINOLOGY
Much of the terminology discussed in this section has been mentioned in the text where appropriate.
s Specific Binding Pair
This describes a pair of molecules (each being a member of a specific binding pair) which are naturally derived or
synthetically produced. One of the pair of molecules, has an area on its surface, or a cavity which specifically binds
to, and is therefore defined as complementary with a particular spatial and polar organisation of the other molecule,
10 so that the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are
antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate, IgG-protein A.
Multimeric Member
15 This describes a first polypeptide which will associate with at least a second polypeptide, when the polypeptides
are expressed in free form and/or on the surface of a substrate. The substrate may be provided by a bacteriophage.
Where there are two associated polypeptides, the associated polypeptide complex is a dimer, where there are three,
a trimer etc. The dimer, trimer, multimer etc or the multimeric member may comprise a member of a specific binding pair.
Example multimeric members are heavy domains based on an immunoglobulin molecule, light domains based on
20 an immunoglobulin molecule, T-cell receptor subunits.
Replicable Genetic Display Package (Rgdp)
This describes a biological particle which has genetic information providing the particle with the ability to replicate.
25 The particle can display on its surface at least part of a polypeptide. The polypeptide can be encoded by genetic
information native to the particle and/or artificially placed into the particle or an ancestor of it. The displayed polypeptide
may be any member of a specific binding pair eg. heavy or light chain domains based on an immunoglobuiin molecule,
an enzyme or a receptor etc.
The particle may be a virus eg. a bacteriophage such as fd or M13.
30
Package
This describes a replicable genetic display package in which the particle is displaying a member of a specific
binding pair at its surface. The package may be a bacteriophage which displays an antigen binding domain at its
35 surface. This type of package has been called a phage antibody (pAb).
Antibody
This describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers
40 any protein having a binding domain which is homologous to an immunoglobulin binding domain. These proteins can
be derived from natural sources, or partly or wholly synthetically produced.
Example antibodies are the immunoglobulin isotypes and the Fab, F(ab 1 ) 2 , scFv, Fv, dAb, Fd fragments.
Immunoglobulin Superfamily
45
This describes a family of polypeptides, the members of which have at least one domain with a structure related
to that of the variable or constant domain of immunoglobulin molecules. The domain contains two (3-sheets and usually
a conserved disulphide bond (see A.F. Williams and A.N. Barclay 1988 Ann. Rev Immunol. 6 381-405).
Example members of an immunoglobulin superfamily are CD4, platelet derived growth factor receptor (PDGFR),
50 intercellular adhesion molecule. (ICAM). Except where the context otherwise dictates, reference to immunoglobulins
and immunoglobulin homologs in this application includes members of the immunoglobulin superfamily and homologs
thereof.
Homologs
55
This term indicates polypeptides having the same or conserved residues at a corresponding position in their pri-
mary, secondary or tertiary structure. The term also extends to two or more nucleotide sequences encoding the ho-
mologous polypeptides.
9
EP0 774 511 A1
Example homologous peptides are the immunoglobulin isotypes.
Functional
5 In relation to a sbp member displayed on the surface of a rgdp, means that the sbp member is presented in a
folded form in which its specific binding domain for its complementary sbp member is the same or closely analogous
to its native configuration, whereby it exhibits similar specificity with respect to the complementary sbp member. In this
respect, it differs from the peptides of Smith at al, supra, which do not have a definite folded configuration and can
assume a variety of configurations determined by the complementary members with which they may be contacted.
10
Genetically diverse population
In connection with sbp members or polypeptide components thereof, this is referring not only to diversity that scan
exist in the natural population of cells or organisms, but also diversity that can be created by artificial mutation in vitro
15 or in vivo .
Mutation in vitro may for example, involve random mutagenesis using oligonucleotides having random mutations
of the sequence desired to be varied. In vivo mutagenesis may for example, use mutator strains of host microorganisms
to harbour the DNA (see Example 38 below).
20 Domain
A domain is a part of a protein that is folded within itself and independently of other parts of the same protein and
independently of a complementary binding member.
25 Folded Unit
This is a specific combination of an a-helix and/or p-strand and/or p-turn structure. Domains and folded units
contain structures that bring together amino acids that are not adjacent in the primary structure.
30 Free Form
This describes the state of a polypeptide which is not displayed by a replicable genetic display package.
Conditionally Defective
35
This describes a gene which does not express a particular polypeptide under one set of conditions, but expresses
it under another set of conditions. An example, is a gene containing an amber mutation expressed in non-suppressing
or suppressing hosts respectively.
Alternatively, a gene may express a protein which is defective under one set of conditions, but not under another
40 set. An example is a gene with a temperature sensitive mutation.
Suppressible Translational Stop Codon
This describes a codon which allows the translation of nucleotide sequences downstream of the codon under one
45 set of conditions, but under another set of conditions translation ends at the codon. Example of suppressible transla-
tional stop codons are the amber, ochre and opal codons.
Mutator Strain
50 This is a host cell which has a genetic defect which causes DNA replicated within it to be mutated with respect to
its parent DNA. Example mutator strains are NR9046mutD5 and NR9046 mut T1 (see Example 38).
Helper Phage
55 This is a phage which is used to infect cells containing a defective phage genome and which functions to comple-
ment the defect. The defective phage genome can be a phagemid or a phage with some function encoding gene
sequences removed. Examples of helper phages are M1 3K07, M1 3K07 gene 1 1 1 no. 3; and phage displaying or encoding
a binding molecule fused to a capsid protein.
10
EP0 774 511 A1
Vector
This is a DNA molecule, capable of replication in a host organism, into which a gene is inserted to construct a
recombinant DNA molecule.
5
Phage Vector
This is a vector derived by modification of a phage genome, containing an origin of replication for a bacteriophage,
but not one for a plasmid.
10
Phagemid Vector
This is a vector derived by modification of a plasmid genome, containing an origin of replication for a bacteriophage
as well as the plasmid origin of replication.
15
Secreted
This describes a rgdp or molecule that associates with the member of a sbp displayed on the rgdp, in which the
sbp member and/or the molecule, have been folded and the package assembled externally to the cellular cytosol.
20
Repertoire of Rearranged Immunoglobulin Genes
A collection of naturally occurring nucleotides eg DNA sequences which encoded expressed immunoglobulin
genes in an animal. The sequences are generated by the in vivo rearrangement of eg V, D and J segments for H chains
25 and eg the V and J segments for L chains. Alternatively the sequences may be generated from a cell line immunised
in vitro and in which the rearrangement in response to immunisation occurs intracellular^.
Library
30 A collection of nucleotide eg DNA, sequences within clones.
Repertoire of Artificially Rearranged Immunoglobulin Genes
A collection of nucleotide eg DNA, sequences derived wholly or partly from a source other than the rearranged
35 immunoglobulin sequences from an animal. This may include for example, DNA sequences encoding VH domains by
combining unrearranged V segments with D and J segments and DNA sequences encoding VL domains by combining
V and J segments.
Part or all of the DNA sequences may be derived by oligonucleotide synthesis.
40 Secretory Leader Peptide
This is a sequence of amino acids joined to the N-terminal end of a polypeptide and which directs movement of
the polypeptide out of the cytosol.
45 Eluant
This is a solution used to breakdown the linkage between two molecules. The linkage can be a non-covalent or
covalent bond(s). The two molecules can be members of a sbp.
so Derivative
This is a substance which derived from a polypeptide which is encoded by the DNA within a selected rgdp. The
derivative polypeptide may differ from the encoded polypeptide by the addition, deletion, substitution or insertion of
amino acids, or by the linkage of other molecules to the encoded polypetide. These changes may be made at the
55 nucleotide or protein level. For example the encoded polypeptide may be a Fab fragment which is then linked to an
Fc tail from another source. Alternatively markers such as enzymes, flouresceins etc may be linked to eg Fab, scFv
fragments.
The present invention provides a method for producing a replicable genetic display package or population such
11
EP0 774 511 A1
rgdps of which method comprises the steps of:
a) inserting a nucleotide sequence encoding a member of a specific binding pair eg. a binding molecule within a
viral genome;
s b) culturing the virus containing said nucleotide sequence, so that said binding molecule is expressed and displayed
by the virus at its surface.
The present invention also provides a method for selecting a rgdp specific for a particular epitope which comprises
producing a population of such rgdps as described above and the additional step of selecting for said binding molecule
10 by contac--ing the population with said epitope so that individual rgdps with the desired specificity may bind to said
epitope. The method may comprise one or more of the additional steps of: (i) separating any bound rgdps from the
epitope; (ii) recovering any separated rgdps and (iii) using the inserted nucleotide sequences from any separated rgdps
in a recombinant system to produce the binding molecule separate from virus. The selection step may isolate the
nucleotide sequence encoding the binding molecule of desired specificity by virtue of said binding molecule being
15 expressed in association with the surface of the virus in which said encoding nucleic acid is contained.
The present invention also provides a method of producing a multimeric member of a specific binding pair (sbp),
which method comprises:
expressing in a recombinant host organism a first polypeptide chain of said sbp member or a genetically diverse pop-
ulation of said sbp member fused to a component of a secreted replicable genetic display package (rgdp) which thereby
20 displays said polypeptide at the surface of the package, and expressing in a recombinant host organism a second
polypeptide chain of said multimer and causing or allowing the polypeptide chains come together to form said multimer
as part of said rgdp at least one of said polypeptide chains being expressed from nucleic acid that is capable of being
packaged using said component therefor, whereby the genetic material of each said rgdp encodes a said polypeptide
chain.
25 Both said chains may be expressed in the same host organism.
The first and second chains of said multimer may be expressed as separate chains from a single vector containing
their respective nucleic acid.
At least one of said polypeptide chains may be expressed from a phage vector.
At least one of said polypeptide chains may be expressed from a phagemid vector, the method including using a
30 helper phage, or a plasmid expressing complementing phage genes, to help package said phagemid genome, and
said component of the rgdp is a capsid protein therefor. The capsid protein may be absent, defective or conditionally
defective in the helper phage.
The method may comprise introducing a vector capable of expressing said first polypeptide chain, into a host
organism which expresses said second polypeptide chain in free form, or introducing a vector capable of expressing
35 said second polypeptide in free form into a host organism which expresses said first polypeptide chain.
Each of the polypeptide chain may be expressed from nucleic acid which is capable of being packaged as a rgdp
using said component fusion product, whereby encoding nucleic acid for both said polypeptide chains are packaged
in respective rgdps.
The nucleic acid encoding at least one of said first and second polypeptide chains may be obtained from a library
40 of nucleic acid including nucleic acid encoding said chain or a population of variants of said chain. Both the first and
second polypeptide chains may be obtained from respective said libraries of nucleic acid.
The present invention also provides a method of producing a member of a specific binding pair (sbp), from a nucleic
acid library including nucleic acid encoding said sbp member or a genetically diverse population of said type of sbp
members, which method comprises:
45 expressing in recombinant host cells polypeptides encoded by said library nucleic acid fused to a component of
a secreted replicable genetic display package (rgdp) or in free form for association with a polypeptide component of
said sbp member which is expressed as a fusion to said rgdp component so that the rgdp displays said sbp member
in functional form at the surface of the package, said library nucleic acid being contained within the host cells in a form
that is capable of being packaged using said rgdp component, whereby the genetic material of an rgdp displaying an
50 sbp member contains nucleic acid encoding said sbp member or a polypeptide component thereof.
The nucleotide sequences for the libraries may be derived from eg animal spleen cells or peripheral blood lym-
phocytes. Alternatively the nucleotide sequence may be derived by the jn vitro mutagenesis of an existing antibody
coding sequence.
The present invention also provides a method of producing a member of a specific binding pair (sbp), which method
55 comprises:
expressing in recombinant host cells nucleic acid encoding said sbp member or a genetically diverse population
of said type of sbp member wherein the or each said sbp member or a polypeptide component thereof is expressed
as a fusion with a component of a secreted replicable genetic display package (rgdp) which displays said sbp member
12
EP0 774 511 A1
at the surface of the package, nucleic acid encoding said sbp member or a polypeptide component thereof being
contained within the host cell in a form that is capable of being packaged using said rgdp component whereby the
genetic material of the rgdp displaying said sbp member encodes said sbp member or a polypeptide component thereof,
said host organism being a mutator strain which introduces genetic diversity into the sbp member to produce said
5 mixed population.
The present invention also provides a method of producing a member of a specific binding pair (sbp), which method
comprises:
expressing in recombinant host cells nucleic acid encoding said sbp member or a genetically diverse population
of said type of sbp member wherein the or each said sbp member or a polypeptide component thereof is expressed
10 as a fusion with a component of a secreted replicable genetic display package (rgdp) which displays said sbp member
in functional form at the surface of the package, nucleic acid encoding said sbp member or a polypeptide component
thereof being contained within the host cell in a form that is capable of being packaged using said rgdp component
whereby the genetic material of the rgdp displaying an sbp member encodes said sbp member or a polypeptide com-
ponent thereof, said fusions being with bacteriophage capsid protein and the rgdps being formed with said fusions in
15 the absence of said capsid expressed in wild-type form.
The present invention also provides a method of producing a member of a specific binding pair (sbp) which method
comprises:
expressing in recombinant host cells nucleic acid encoding said sbp member or a genetically diverse population
of said type of sbp member or a polypeptide component thereof fused to a component of a secreted replicable genetic
20 display package (rgdp) which displays said sbp member in functional form at the surface of the package, nucleic acid
encoding said sbp member or a polypeptide component thereof being contained within the host cell in a form that is
capable of being packaged using said rgdp component whereby the genetic material of the rgdp displaying an sbp
member or a polypeptide component thereof encodes said sbp member or a polypeptide component thereof, said sbp
member or polypeptide component thereof being expressed from a phagemid as a capsid fusion, and a helper phage,
25 or a plasmid expressing complementing phage genes, is used along with said capsid fusions to package the phagemid
nucleic acid.
The library or genetically diverse population may be obtained from:
(i) the repertoire of rearranged immunoglobulin genes of an animal immunised with complementary sbp member,
30 (jj) the repertoire of rearranged immunoglobulin genes of an animal not immunised with complementary sbp mem-
ber,
(iii) a repertoire of artificially rearranged immunoglobulin gene or genes
(iv) a repertoire of immunoglobulin homolog gene or genes; or
(v) mixture of any of (i), (ii), (iii) and (iv).
35
The capsid protein may be absent, defective or conditionally defective in the helper phage.
The host cell may be a mutator strain which introduces genetic diversity into the sbp member nucleic acid.
The sbp member may comprise a domain which is, or is homologous to, an immunoglobulin domain.
The rgdp may be a bacteriophage, the host a bacterium, and said component of the rgdp a capsid protein for the
40 bacterophage. The phage may be a filamentous phage. The phage may be selected from the class I phages fd, M13,
f1 , If1 , Ike, ZJ/Z, Ff and the class II phages Xf, Pf1 and Pf3. The phage may be fd or a derivative of fd. The derivative
may be tetracycline resistant. The said sbp member or polypeptide chain thereof may be expressed as a fusion with
the gene III capsid protein of phage fd or its counterpart in another filamentous phage. The sbp member or polypeptide
chain thereof may be inserted in the N-terminal region of the mature capsid protein downstream of a secretory leader
45 peptide. The sequence may be inserted after amino acid +1 of the mature protein. The site for insertion may be flanked
by short sequences corresponding to sequences which occur at each end of the nucleic acid to be inserted. For example
where 4 the protein domain is an immunoglobulin domain, the insertion site in the phage may be flanked by nucleotide
sequences which code for the first five amino acids and the last five amino acids of the Ig domain. Such flanking
nucleotide sequences are shown in figure 4(2) B and C, wherein the site-flanking nucleotide sequences encode amino
50 acid sequences QVQLQ and VTVSS which occur at either end of the VH domain, or QVQLQ and LEIKR which occur
at either end of the Fv (combined VH + VL) domain. Each of these sequences flanking the insertion site may include
a suitable cleavage site, as shown in Fig 4.
Alternatively, the flanking nucleotide sequences shown in figure 4(2)B and C as described above, may be used to
flank the insertion site for any nucleic acid to be inserted, whether or not that nucleic acid codes an immunoglobulin.
55 The host may be E.coli.
Nucleic acid encoding an sbp member polypeptide may be linked downstream to a viral capsid protein through a
suppressible translational stop codon.
As previously mentioned, the present invention also provides novel selection systems and assay formats. In these
13
EP0 774 511 A1
systems and formats, the gene sequence encoding the binding molecule (eg, the antibody) of desired specificity is
separated from a general population of rgdps having a range of specifies, by the fact of its binding to a specific target
(eg the antigen or epitope). Thus the rgdps formed by said expression may be selected or screened to provide an
individual sbp member or a selected mixed population of said sbp members associated in their respective rgdps with
s nucleic acid encoding said sbp member or a polypeptide chain thereof. The rgdps may be selected by affinity with a
member complementary to said sbp member.
Any rgdps bound to said second member may be recovered by washing with an eluant. The washing conditions
may be varied in order to obtain rgdps with different binding affinities for said epitope. Alternatively, to obtain eg high
affinity rgdps, the complementary member (eg an epitope) may be presented to the population of rgdps (eg pAbs)
10 already bound to a binding member in which case pAbs with a higher affinity for the epitope will displace the already
bound binding member. Thus the eluant may contain a molecule which competes with said rgdp for binding to the
complementary sbp member. The rgdp may be applied to said complementary sbp member in the presence of a mol-
ecule which competes with said package for binding to said complementary sbp member. Nucleic acid derived from a
selected or screened rgdp may be used to express said sbp member or a fragment or derivative thereof in a recombinant
15 host organism. Nucleic acid from one or more rgdps may be taken and used to provide encoding nucleic acid in a
further said method to obtain an individual sbp member or a mixed population of sbp members, or encoding nucleic
acid therefor. The expression end product may be modified to produce a derivative thereof.
The expression end product or derivative thereof may be used to prepare a therapeutic or prophylactic medicament
or a diagnestic product.
20 The present invention also provides recombinant host cells harbouring a library of nucleic acid fragments com-
prising fragments encoding a genetically diverse population of a type of member of a specific binding pair (sbp), each
sbp member or a polypeptide component thereof being expressed as a fusion with a component of a secretable rep-
licable genetic display package (rgdp), so that said sbp members are displayed on the surface of the rgdps in functional
form and the genetic material of the rgdps encode the associated sbp member or a polypeptide component thereof.
25 The type of sbp members may be immunoglobulins or immunoglobulin homologs, a first polypeptide chain of which is
expressed as a said fusion with a component of the rgdp and a second polypeptide chain of which is expressed in free
form and associates with the fused first polypeptide chain in the rgdp.
The present invention also provides a helper phage whose genome lacks nucleic acid encoding one of its capsid
proteins, or whose encoding nucleic acid therefor is conditionally defective, or which encodes said capsid protein in
30 defective or conditionally defective form.
The present invention also provides a bacterial host cell containing a filamentous phage genome defective for a
capsid protein thereof and wherein the host cell is capable of expressing capsid protein complementing said defect
such that infectious phage particles can be obtained therefrom. The complementing capsid protein may be expressed
in said host from another vector contained therein. The defective capsid protein may be gene III of phage fd or its
35 counterpart in another filamentous phage.
The present invention also provides recombinant E.coli TG1 M13K07 gill No. 3 (NCTC 12478).
The present invention also provides a phage antibody having the form of a replicable genetic display package
displaying on its surface in functional form a member of a specific binding pair or a specific binding domain thereof.
In the above methods, the binding molecule may be an antibody or a domain that is homologous to an immu-
40 noglobulin. The antibody and/or domain may be either naturally derived or synthetic or a combination of both. The
domain may be a Fab, scFv, Fv dAb or Fd molecule. Alternatively, the binding molecule may be an enzyme or receptor
or fragment, derivative or analogue of any such enzyme or receptor. Alternatively, the binding molecule may be a
member of an immunoglobulin superfamily and which has a structural form based on an immunoglobulin molecule.
The present invention also provides rgdps as defined above and members of specific binding pairs eg. binding
45 molecules such as antibodies, enzymes, receptors, fragments and derivatives thereof, obtainable by use of any of the
above defined methods. The derivatives may comprise members of the specific binding pairs fused to another molecule
such as an enzyme or a Fc tail.
The invention also includes kits for carrying out the methods hereof. The kits will include the necessary vectors.
One such vector will typically have an origin of replication for single stranded bacteriophage and either contain the sbp
50 member nucleic acid or have a restriction site for its insertion in the 5' end region of the mature coding sequence of a
phage capsid protein, and with a secretory leader coding sequence upstream of said site which directs a fusion of the
capsid protein exogenous polypeptide to the periplasmic space.
The restriction sites in the vectors are preferably those of enzymes which cut only rarely in protein coding sequenc-
es.
55 The kit preferably includes a phagemid vector which may have the above characteristics, or may contain, or have
a site for insertion, of sbp member nucleic acid for expression of the encoded polypeptide in free form.
The kits will also contain ancillary components required for carrying out the method, the nature of such components
depending of course on the particular method employed.
14
EP0 774 511 A1
Useful ancillary components may comprise helper phage, PCR primers, and buffers and enzymes of various kinds.
PCR primers and associated reagents for use where the sbp members are antibodies may have the following
characteristics:
s (i) primers having homology to the 5' end of the sense or anti-sense strand of sequences encoding domains of
antibodies; and
(ii) primers including tag sequences 5' to these homologous sequences which incorporate restriction sites to allow
insertion into vectors; together with sequences to allow assembly of amplified VH and VL regions to enable ex-
pression as Fv, scFv or Fab fragments.
w
Buffers and enzymes are typically used to enable preparation of nucleotide sequences encoding Fv, scFv or Fab
fragments derived from rearranged or unrearranged immunoglobulin genes according to the strategies described here-
in.
The applicants have chosen the filamentous F-specific bacteriophages as an example of the type of phage which
15 could provide a vehicle for the display of binding molecules e.g. antibodies and antibody fragments and derivatives
thereof, on their surface and facilitate subsequent selection and manipulation.
The F-specific phages (e.g. f1, fd and M13) have evolved a method of propagation which does not kill the host
cell and they are used commonly as vehicles for recombinant DNA (Kornberg, A., DNA Replication, W.H. Freeman
and Co., San Francisco, 1980). The single stranded DNA genome (approximately 6.4 Kb) of fd is extruded through
20 the bacterial membrane where it sequesters capsid sub-units, to produce mature virions. These virions are 6 nm in
diameter, "Iujti in length and each contain approximately 2,800 molecules of the major coat protein encoded by viral
gene VIII and four molecules of the adsorption molecule gene III protein (g3p) the latter is located at one end of the
virion. The structure has been reviewed by Webster at al., 1 978 in The Single Stranded DNA Phages, 557-569, Cold
Spring Harbor Laboratory Press. The gene III product is involved in the binding of the phage to the bacterial F-pilus.
25 Although these phages do not kill their host during normal replication, disruption of some of their genes can lead
to cell death (Kornberg, A., 1 980 supra.) This places some restraint on their use. The applicants have recognized that
gene III of phage fd is an attractive possibility for the insertion of biologically active foreign sequences. There are
however, other candidate sites including for example gene VIII and gene VI.
The protein itself is only a minor component of the phage coat and disruption of the gene does not lead to cell
30 death (Smith, G. 1988, Virology 167 : 156-165). Furthermore, it is possible to insert some foreign sequences (with no
biological function) into various positions within this gene (Smith, G. 1985 Science 228 : 1315-1317., Parmley, S.F. and
Smith, G.P. Gene: 73 (1988) p. 305-318., and de la Cruz, V.F., at al., 1988, J. Biol. Chem., 263: 4318-4322). Smith at
al described the display of peptides on the outer surface of phage but they did not describe the display of protein
domains. Peptides can adopt a range of structures which can be different when in free solution, than when bound to,
35 for example, an antibody, or when forming part of a protein (Stanfield, R.I. at al., (1990) Science 248 , p712-719).
Proteins in general have a well defined tertiary structure and perform their biological function only when adopting this
structure. For example, the structure of the antibody D1 .3 has been solved in the free form and when bound to antigen
(Bhat, T.N.. at al., (1990) Nature 347, p483-485). The gross structure of the protein is identical in each instance with
only minor variations around the binding site for the antigen. Other proteins have more substantial conformation chang-
40 es on binding of ligand, for instance the enzymes hexokinase and pyruvate dehydrogenase during their catalytic cycle,
but they still retain their overall pattern of folding. This structural integrity is not confined to whole proteins, but is
exhibited by protein domains. This leads to the concept of a folded unit which is part of a protein, often a domain, which
has a well defined primary, secondary and tertiary structure and which retains the same overall folding pattern whether
binding to a binding partner or not. The only gene sequence that Smith at al., described that was of sufficient size to
45 encode a domain (a minimum of perhaps 50 amino acids) was a 335bp fragment of a p-galctrosidase corresponding
to nucleotides 861-1195 in the p-galactosidase gene sequence (Parmley, S. + Smith, G.P. 1988 supra. This would
encode 112 amino acids of a much larger 380 amino acid domain. Therefore, prior to the present application, no
substantially complete domain or folded unit had been displayed on phage. In these cases, although the infectivity of
the virion was disrupted, the inserted sequences could be detected on the phage surface by use of e.g. antibodies.
50 The protein encoded by gene III has several domains (Pratt, D., at al., 1 969 Virology 39:42-53., Grant, R.A., et al.,
1981, J. Biol. Chem. 256: 539-546 and Armstrong, J., et al., FEBS Lett. T35: 167-172 1981 .) including: (i) a signal
sequence that directs the protein to the cell membrane and which is then cleaved off; (ii) a domain that anchors the
mature protein into the bacterial cell membrane (and also the phage coat); and (iii) a domain that specifically binds to
the phage receptor, the F-pilus of the host bacterium. Short sequences derived from protein molecules have been
55 inserted into two places within the mature molecule (Smith, G., 1985 supra., and Parmley, S.F. and Smith G.P, 1988
supra.). Namely, into an inter-domain region and also between amino acids 2 and 3 at the N-terminus. The insertion
sites at the N-terminus were more successful in maintaining the structural integrity of the gene III protein and displaying
the peptides on the surface of the phage. By use of antisera specific for the peptides, the peptides inserted into this
15
EP0 774 511 A1
position were shown to be on the surface of the phage. These authors were also able to purify the phage, using this
property. However, the peptides expressed by the phage, did not possess measurable biological functions of their own.
Retaining the biological function of a molecule when it is expressed in a radically different context to its natural
state is difficult. The demands on the structure of the molecule are heavy. In contrast, retaining the ability to be bound
5 by specific antisera is a passive process which imposes far less rigorous demands on the structure of the molecule.
For example, it is the rule rather than the exception that polyclonal antisera will recognise totally denatured, and bio-
logically inactive, proteins on Western blots (see for example, Harlow, E. and Lane, D., Antibodies, a Laboratory Manual,
Cold Spring Haroor Laboratory Press 1 988). Therefore, the insertion of peptides into a region that allows their structure
to be probed with antisera teaches only that the region allows the inserted sequences to be exposed and does not
10 teach that the region is suitable for the insertion of large sequences with demanding structural constraints for the display
of a molecule with a biological or binding function. In particular, it does not teach that domains or folded units of proteins
can be displayed from sequences inserted in this region.
This experience with Western blots is a graphic practical demonstration which shows that retaining the ability to
be bound by specific antisera imposes far less rigorous demands on the structure of a polypeptide, than does folding
15 for the retention of a biological function.
Studies have been carried out, in which E.coli have been manipulated to express the protein p-adrenergic receptor
as a fusion with the outer membrane protein lamB. The p-adrenergic receptor was expressed in a functional form as
determined by the presence of binding activity. However, when an equivalent antibody fusion was made with lamB,
the antibody fusion was toxic to the host cell.
20 The applicants have investigated the possibility of Inserting the gene coding sequence for biologically active an-
tibody fragments into the gene III region of fd to express a large fusion protein. As is apparent from the previous
discussion, this approach makes onerous demands on the functionality of the fusion protein. The insertion is large,
encoding antibody fragments of at least 100-200 amino acids; the antibody derived domain must fold efficiently and
correctly to display antigen-binding; and most of the functions of gene III must be retained. The applicants approach
25 to the construction of the fusion molecule was designed to minimise the risk of disrupting these functions. In an em-
bodiment of the invention, the initial vector used was fd-tet (Zacher, A.N., at al., 1 980, Gene 9, 127-140) a tetracycline
resistant version of fd bacteriophage that can be propagated as a plasmid that confers tetracycline resistance to the
infected E.coli host. The applicants chose to insert after the signal sequence of the fd gene III protein for several
reasons. In particular, the applicants chose to insert after amino acid 1 of the mature protein to retain the context for
30 the signal peptidase cleavage. To retain the structure and function of gene III itself, the majority of the original amino
acids are synthesized after the inserted immunoglobulin sequences. The inserted immunoglobulin sequences were
designed to include residues from the switch region that links VH-VL to CH1-CL (Lesk, A., and Chothia, C, Nature
335, 188-190, 1988).
Surprisingly, by manipulating gene III of bacteriophage fd, the present applicants have been able to construct a
35 bacteriophage that displays on its surface large biologically functional antibody, enzyme, and receptor molecules whilst
remaining intact and infectious. Furthermore, the phages bearing antibodies of desired specificity, can be selected
from a background of phages not showing this specificity.
The sequences coding for a population of antibody molecules and for insertion into the vector to give expression
of antibody binding functions on the phage surface can be derived from a variety of sources. For example, immunised
40 or non-immunised rodents or humans, and from organs such as spleen and peripheral blood lymphocytes. The coding
sequences are derived from these sources by techniques familiar to those skilled in the art (Orlandi, R., et al., 1989
supra; Larrick, J.W., etal., 1989 supra; Chiang, Y.L., etal., 1989 Bio Techniques 7, p. 360-366; Ward, E.S, etal., 1989
supra; Sastry, L, et al., 1989 supra.) or by novel linkage strategies described in examples 14, 33, 40 and 42. Novel
strategies are described in examples 7, 25, 33, 39 and 40 for displaying dimeric molecules eg Fab and Fv fragments
45 on the surface of a phage. Each individual pAb in the resulting library of pAbs will express-antibodies or antibody
derived fragments that are monocional with respect to their antigen-binding characteristics.
The disclosure made by the present applicants is important and provides a significant breakthrough in the tech-
nology relating to the production of biological binding molecules, their fragments and derivatives by the use of recom-
binant methods.
50 in standard recombinant techniques for the production of antibodies, an expression vector containing sequences
coding for the antibody polypeptide chains is used to transform e.g. E.coli. The antibody polypeptides are expressed
and detected by use of standard screening systems. When the screen detects an antibody polypeptide of the desired
specificity, one has to return to the particular transformed E.coli expressing the desired antibody polypeptide. Further-
more, the vector containing the coding sequence for the desired antibody polypeptide then has to be isolated for use
55 from E.coli in further processing steps.
In the present invention however, the desired antibody polypeptide when expressed, is already packaged with its
gene coding sequence. This means that when the an antibody polypeptide of desired specificity is selected, there is
no need to return to the original culture for isolation of that sequence. Furthermore, in previous methods in standard
16
EP0 774 511 A1
recombinant techniques, each clone expressing antibody needs to be screened individually. The present application
provides for the selection of clones expressing antibodies with desired properties and thus only requires screening of
clones from an enriched pool.
Because a rgdp (eg a pAb) is a novel structure that displays a member of a specific binding pair (eg. an antibody
5 of monoclonal antigen-binding specificity) at the surface of a relatively simple replicable structure also containing the
genetic information encoding the member, rgdps eg pAbs, that bind to the complementary member of the specific
binding pair (eg antigen) can be recovered very efficiently by either eluting off the complementary member using for
example diethylamine, high salt etc and infecting suitable bacteria, or by denaturing the structure, and specifically
amplifying the sequences encoding the member using PCR. That is, there is no necessity to refer back to the original
10 bacterial clone that gave rise to the pAb.
For some purposes, for example immunoprecipitation and some diagnostic tests, it is advantageous to use poly-
clonal antibodies or antibody fragments. The present invention allows this to be achieved by either selection of an
enriched pool of pAbs with desired properties or by mixing individually isolated clones with desired properties. The
antibodies or antibody fragments may then be expressed in soluble form if desired. Such a selected polyclonal pAb
15 population can be grown from stocks of phage, bacteria containing phagemids or bacteria expressing soluble fragments
derived from the selected polyclonal population. Thus a reagent equivalent to a polyclonal antiserum is created which
can be replicated and routinely manufactured in culture without use of animals.
SELECTION FORMATS AND AFFINITY MATURATION
20
Individual rgdps eg pAbs expressing the desired specificity eg for an antigen, can be isolated from the complex
library using the conventional screening techniques (e.g. as described in Harlow, E., and Lane, D., 1 988, supra Gher-
ardi, E at al. 1 990. J. Immunol, math. T26 p61 -68).
The applicants have also devised a series of novel selection techniques that are practicable only because of the
25 unique properties of rgdps. The general outline of some screening procedures is illustrated in figure 2 using pAbs as
an example type of rgdp.
The population/library of pAbs to be screened could be generated from immunised or other animals; or be created
in vitro by mutagenising pre-existing phage antibodies (using techniques well-known in the art such as oligonucleotide
directed mutagenesis (Sambrook, J., at al., 1989 Molecular Cloning a Laboratory Manual, Cold Spring Harbor Labo-
30 ratory Press). This population can be screened in one or more of the formats described below with reference to figure
2, to derive those individual pAbs whose antigen binding properties are different from sample c.
Binding Elution
35 Figure 2(i) shows antigen (ag) bound to a solid surface (s) the solid surface (s) may be provided by a petri dish,
chromatography beads, magnetic beads and the like. The population/library of pAbs is then passed over the ag, and
those individuals p that bind are retained after washing, and optionally detected with detection system d. A detection
system based upon anti-fd antisera is illustrated in more detail below in example 4. If samples of bound population p
are removed under increasingly stringent conditions, the binding affinity represented in each sample will increase.
40 Conditions of increased stringency can be obtained, for example, by increasing the time of soaking or changing the
pH of the soak solution, etc.
Competition
45 Referring to figure 2(H) antigen ag can be bound to a solid support s and bound to saturation by the original binding
molecule c. If a population of mutant pAb (or a set of unrelated pAbs) is offered to the complex, only those that have
higher affinity for antigen ag than c will bind. In-most examples, only a minority of population c will be displaced by
individuals from population p. If c is a traditional antibody molecule, all bound material can be recovered and bound p
recovered by infecting suitable bacteria and/or by use of standard techniques such as PCR.
50 An advantageous application is where ag is used as a receptor and c the corresponding ligand. The recovered
bound population p is then related structurally to the receptor binding site/and or ligand. This type of specificity is known
to be very useful in the pharmaceutical industry.
Another advantageous application is where ag is an antibody and c its antigen. The recovered bound population
p is then an anti-idiotype antibody which have numerous uses in research and the diagnostic and pharmaceutical
55 industries.
At present it is difficult to select directly for anti-idiotype antibodies. pAbs would give the ability to do this directly
by binding pAb libraries (eg a naive library) to B cells (which express antibodies on their surface) and isolating those
phage that bound well.
17
EP0 774 511 A1
In some instances it may prove advantageous to preselect population p. For example, in the anti-idiotype example
above, p can be absorbed against a related antibody that does not bind the antigen.
However, if c is a pAb, then either or both c and p can advantageously be marked in some way to both distinguish
and select for bound p over bound c. This marking can be physical, for example, by pre-labelling p with biotin; or more
5 advantageously, genetic. For example, c can be marked with an EcoB restriction site, whilst p can be marked with an
EcoK restriction site (see Carter, P. at al., 1985, Nucl. Acids Res. J_3, 4431-4443). When bound p+c are eluted from
the antigen and used to infect suitable bacteria, there is restriction (and thus no growth) of population c (i.e. EcoB
restricting bacteria in this example). Any phage that grew, would be greatly enriched for those individuals from p with
higher binding affinities. Alternatively, the genetic marking can be achieved by marking p with new sequences, which
10 can be used to specifically amplify p from the mixture using PCR.
Since the bound pAbs can be amplified using for example PCR or bacterial infection, it is also possible to rescue
the desired specificity even when insufficient individuals are bound to allow detection via conventional techniques.
The preferred method for selection of a phage displaying a protein molecule with a desired specificity or affinity
will often be elution from an affinity matrix with a ligand (eg example 21). Elution with increasing concentrations of
15 ligand should elute phage displaying binding molecules of increasing affinity. However, when eg a pAb binds to its
antigen with high affinity or avidity (or another protein to its binding partner) it may not be possible to elute the pAb
from an affinity matrix with molecule related to the antigen. Alternatively there may be no suitable specific eluting
molecule that can be prepared in sufficiently high concentration. In these cases it is necessary to use an elution method
which is not specific to eg the antigen-antibody complex. Some of the non-specific elution methods generally used
20 reduce phage viability for instance, phage viability is reduced with time at pH12 (Rossomando, E.F. and Zinder N.D.
J. Mol.Biol. 36 387-399 1968). There may be interactions between eg antibodies and affinity matrices which cannot
be disrupted without completely removing phage infectivity. In these cases a method is required to elute phage which
does not rely on disruption of eg the antibody - antigen interaction. A method was therefore devised which allows
elution of bound pAbs under mild conditions (reduction of a dithiol group with dithiothreitol) which do not disrupt phage
25 structure (example 47).
This elution procedure is just one example of an elution procedure under mild conditions. A particularly advanta-
geous method would be to introduce a nucleotide sequence encoding amino acids constituting a recognition site for
cleavage by a highly specific protease between the foreign gene inserted, in this instance a gene for an antibody
fragment, and the sequence of the remainder of gene III. Examples of such highly specific proteases are Factor X and
30 thrombin. After binding of the phage to an affinity matrix and elution to remove non-specific binding phage and weak
binding phage, the strongly bound phage would be removed by washing the column with protease under conditions
suitable for digestion at the cleavage site. This would cleave the antibody fragment from the phage particle eluting the
phage. These phage would be expected to be infective, since the only protease site should be the one specifically
introduced. Strongly binding phage could then be recovered by infecting eg. E.coli TG1 cells.
35 An alternative procedure to the above is to take the affinity matrix which has retained the strongly bound pAb and
extract the DNA, for example by boiling in SDS solution. Extracted DNA can then be used to directly transform E.coli
host cells or alternatively the antibody encoding sequences can be amplified, for example using PCR with suitable
primers such as those disclosed herein, and then inserted into a vector for expression as a soluble antibody for further
study or a pAb for further rounds of selection.
40 Another preferred method for selection according to affinity would be by binding to an affinity matrix containing low
amounts of ligand.
If one wishes to select from a population of phages displaying a protein molecule with a high affinity for its ligand,
a preferred strategy is to bind a population of phage to an affinity matrix which contains a low amount of ligand. There
is competition between phage, displaying high affinity and low affinity proteins, for binding to the ligand on the matrix.
45 Phage displaying high affinity protein is preferentially bound and low affinity protein is washed away. The high affinity
protein is then recovered by elution with the ligand or by other procedures which elute the phage from the affinity matrix
(example 35 demonstrates this procedure).
In summary then, for recovery of the packaged DNA from the affinity step, the package can be simply eluted, it
can be eluted in the presence of a homologous sbp member which competes with said package for binding to a com-
50 plementary sbp member; it could be removed by boiling, it could be removed by proteolytic cleavage of the protein;
and other methods will be apparent to those skilled in the art eg. destroying the link between the substrate and com-
plementary sbp member to release said packaged DNA and sbp member. At any rate, the objective is to obtain the
DNA from the package so that it can be used directly or indirectly, to express the sbp member encoded thereby.
The efficiency of this selection procedure for pAbs and the ability to create very large libraries means that the
55 immunisation techniques developed to increase the proportion of screened cells producing antibodies of interest will
not be an absolute requirement. The technique allows the rapid isolation of binding specificities eg antigen-binding
specificities, including those that would be difficult or even unobtainable by conventional techniques, for example,
catalytic or anti-idiotypic antibodies. Removal of the animal altogether is now possible, once a complete library of the
18
EP0 774 511 A1
immune repertoire has been constructed.
The novel structure of the pAb molecule can be used in a number of other applications, some examples of which are:
Signal Amplification
5
Acting as a novel molecular entity in itself, rgdps eg pAbs combine the ability to bind a specific molecule eg antigen
with amplification, if the major coat protein is used to attach another moiety. This moiety can be attached via immuno-
logical, chemical, or any other means and can be used, for example, to label the complex with detection reagents or
cytotoxic molecules for use jn vivo or jn vitro .
10
Physical Detection
The size of the rgdps eg pAbs can be used as a marker particularly with respect to physical methods of detection
such as electron microscopy and/or some biosensors, e.g. surface plasmon resonance.
15
Diagnostic Assays
The rgdps eg pAbs also have advantageous uses in diagnostic assays, particularly where separation can be ef-
fected using their physical properties for example centrifugation, filtration etc.
20 in order that the invention is more fully understood, embodiments will now be described in more detail by way of
example only and not by way of limitation with reference to the figures described below.
Figure 1 shows the basic structure of the simplest antibody molecule IgG.
Figure 2 shows schematically selection techniques which utilise the unique properties of pAbs; 2i) shows a binding/
elution system; and (2ii) shows a competition system (p=pAb; ag=antigen to which binding by pAb is required; c=com-
25 petitor population e.g. antibody, pAb, ligand; s=substrate (e.g. plastic beads etc); d=detection system.
Figure 3 shows the vector fd-tet and a scheme for the construction of vectors, fdTPs/Bs (for insertion of VH coding
sequences) and fdTPs/Xh for the insertion of scFv coding sequences.
Figure 4 shows the nucleotide sequences for the oligonucleotides and vectors. All sequences are drawn 5' to 3'
and are numbered according to Beck at al., 1978, Nucl. Acid Res., 5: 4495-4503. 4.1 shows the sequences of the
30 oligonucleotides used for mutagenesis (oligo's 1 and 2) or sequencing (oligo 3). The sequences shown were synthe-
sized on an Applied Biosystems, oligonucleotide synthesizer and are complementary to the single stranded form of
fd-tet (they are in the anti-sense form with respect to gene III). 4.2 shows the sequences of the various constructs
around the gene III insertion site. These sequences are drawn in the sense orientation with respect to gene III; (A) fd-
tet (and fdT5Bst) (B) fdTPs/Bs and (C) fdTPs/Xh. The key restriction enzyme sites are shown along with the immu-
35 noglobulin amino acids contributed by the vectors, (amino acid single letter code is used, see Harlow, E., and Lane,
D., 1988 supra.).
Figure 5 shows the nucleotide and amino acid sequences for scFv in the vector scFvD1.3 myc. This gives the
sequence of the anti-lysozyme single chain Fv and surrounding sequences in scFvDI .3 myc, showing the N-terminal
pel B signal peptide sequence and the C-terminal myc tag sequence (Ward, E.S., at al., 1989, supra.). Also shown is
40 the peptide sequence linking the VH and VL regions. The amino acid sequence is represented above the nucleotide
sequence by the single letter code, see Harlow, E., and Lane D., 1 988 supra.
Figure 6 shows the binding of pAbs to lysozyme and the effect of varying the amount of supernatant. Each point
is the average of duplicate samples. Lysozyme was coated at 1 mg/ml in 50 mM NaHC0 3 .
Figure 7 shows the effect of varying the coating concentration of lysozyme or bovine serum albumin on the binding
45 of pAbs to lysozyme in graphical form. Each point is the average of duplicate samples.
Figure 8 shows the sequence around the cloning site in gene III of fd-CAT2. Restriction enzyme sites are shown,
as well as the amino acids encoded by antibody derived sequences. These are flanked at the 5' end by the gene III
signal peptide and at the 3' end by 3 alanine residues (encoded by the Not 1 restriction site) and the remainder of the
mature gene III protein. The arrow shows the cleavage site for cutting of the signal peptide.
so Figure 9 shows the binding of pAb (1 .3) to lysozymes. Binding of phage as detected by ELISAto (a) hen egg-white
lysozyme (HEL) (b) turkey egg-white lysozyme (TEL), (c) human lysozyme (HUL), (d) bovine serum albumin (BSA). A
further control of (e) fdTPs/Bs to HEL.
Figure 1 0 shows a map of FabD1 .3 in pUC1 9.
Figure 11 shows the ELISA results providing a comparison of lysozyme-binding by phage-Fab and phage-scFv.
55 Vector=fdCAT2 (example 5); fdscFv(Ox)=pAbNQ11 (Example 9); fdVHCHI (D1.3)=grown in normal cells (i.e. no L
chain, see example 7); fdFab(D1.3) i.e. fdVHCHI (D1.3) grown in cells containing D1.3 L chain; fdscFv (D1.3)
=pAbD1.3.
Figure 12 shows oligonucleotide probing of affinity purified phage. 10 12 phage in the ratio of 1 pAb (D1.3) in 4 x
19
EP0 774 511 A1
10 4 fdTPS/Bs phages were affinity purified and probed with an oligonucleotide specific for pAb (D1 .3) A is a filter after
one round of affinity purification (900 colonies total) and B is a filter after two rounds (372 colonies total).
Figure 1 3 shows the sequence of the anti-oxazolone antibody fragment NQ1 1 scFv. The sequence contributed by
the linker is shown in the lower case. The sequence for VH is before the linker sequence and the sequence for VL is
5 after the linker.
Figure 14 shows the ELISA results for binding pAb NQ11 and pAb D1 .3 and vector fdTPs/xh to specified antigens.
Figure 15 shows the sequence surrounding the phoA insertion in fd-phoAla166. The restriction sites used for
cloning are shown, as well as the amino acids encoded by phoA around the insertion site. The first five amino acids
of the mature fusion come from gene III.
10 Figure 16(1) shows the structure of gene III and the native BamHI site into which a scFv coding sequence was
inserted in example 13 and figure 16(2) shows the natural peptide linker sites A and B for possible insertion of scFv
coding sequences.
Figure 1 7 shows schematically the protocol for PCR assembly of mouse VH and VLK repertoires for phage display
described in example 14.
15 Figure 18 shows examples of the final products obtained with the procedure of example 14. Lanes a and b show
the products of the initial PCR using heavy and light chain primers respectively; lane c shows the complete assembled
700bp product before final digestion with Not1 and Apal_1 ; M1, M2 markers <E>174 Hae III digest and 123 base pair
ladder (BRL Limited, P.O. Box 35, Washington Road, Paisley, Scotland) respectively.
Figure 1 9 shows the binding of 125 I-PDGF-BB to fd h-PDGFB-R phage in immunoprecipitation assay and compar-
20 json to fdTPs/Bs and no phage controls; binding is expressed as a percentage of the total 125 l-PDGF-BB added to
the incubation.
Figure 20 shows the displacement of 125 I-PDGF-BB bound to fd-h-PDGFB-R phage using unlabelled PDGF-BB
measured using an immunoprecipitation assay. Binding is expressed as a percentage of the total 125 l -PDGF-BB added
to the incubation.
25 Figure 21 shows the displacement of 125 I-PDGF-BB bound to fd-h-PDGFB-R phage using unlabelled PDGF-BB
measured using an immunoprecipitation assay. Non-specific binding of 125 l-PDGF-BBto vector phage fdTPs/Bs in the
absence of added unlabelled PDGF was deducted from each point.
Figure 22 shows the results of an ELISA of lysozyme binding by pCAT-3 scFv D1 .3 phagemid in comparison with
pCAT-3 vector (both rescued by M1 3K07) andfdCAT2 scFv D1 .3 as described in example 17. The ELISA was performed
30 as described in example 6 with modifications detailed in example 18.
Figure 23 shows the digestion pattern seen when individual clones, selected at random from a library of single
chain Fv antibody genes derived from an immunised mouse; are digested with BstNI.
Figure 24 shows VH and VK gene sequences derived from the combinatorial library in example 21 and the hier-
archical library in example 22.
35 Figure 25 shows a matrix of ELISA signals for clones derived from random combinatorial library. Designation of
the clones is as in figure 24. The number of clones found with each combination is shown by the numerals.
Figure 26 shows a) the phagemid pHEN1 a derivative of pUC11 9 described in example 24; and b) the cloning sites
in the phagemid pHEN.
Figure 27. The antibody constructs cloned into fd-CAT2 and pHEN1 for display on the surface of phage. Constructs
40 |, M, in and IV were cloned into both fd-CAT2 (as ApaLI-Notl fragments) and pHEN1 (as Sfil-Notl fragments) and pHEN1
(as Sfil-Notl fragments). All the constructs contained the-heavy chain (VH) and light chain (VK) variable regions of the
mouse anti-phOx antibody NQ10.12.5. The constant domains were human CK and CH1 (y 1 isotype).
Figure 28. Three ways of displaying antibody fragments on the surface of phage by fusion to gene III protein.
Figure 29. Western blot of supernatant taken from pHEN1-ll(+) or pHEN1(-) cultures in E.coli HB2151, showing
45 secretion of Fab fragment from pHEN 1 -II only. The anti-human Fab detects both H and L chain. Due to the attached
c-myc tag, the L chain, highlighted by both anti-c-myc tag and anti-human CK antisera, is slightly larger (calculated Mr
24625) than the H chain (calculated Mr23145).
Figure 30 is a plot showing the effect of lysozyme dilution on ratio of ELISA signals obtained using pAbD1.3 or
soluble scFv D1 .3.
50 Figure 31 is a plot showing the effect of lysozyme dilution on ELISA signals obtained using fdTscFvDI .3 and soluble
scFvD1.3.
Figure 32 is a plot showing positive results from an ELISA screen of phage displaying scFv fragments derived
from the cell line 013 which express a monoclonal antibody directed against oestriol.
Figure 33 is a plot showing positive results from an ELISA screen of phage displaying scFv fragments derived
55 from the cell line 014 which express a monoclonal antibody directed against oestriol.
Figure 34 is a Western Blot showing expression of the alkaline phosphatase-gene 3 fusion. 16uJ of 50 fold con-
centrate of each phage sample was detected on western blots with either anti-gene 3 antiserum (e-f) or with anti-
alkaline phosphatase antiserum (c-f)
20
EP0 774 511 A1
a) fd-phoAla166 grown in TG1 cells
b) fd-phoAla166 grown in KS272 cells
c) fdCCAT2 grown in TG1 cells
d) fdCAT2 grown in TG1 cells, mixed with 13 ng of purified alkaline phosphatase
5 e) fd-phoAla166 grown in TG1 cells
f) fdCAT2 grown in TG1 cells.
Figure 35 is a Western Blot showing ultrafiltration of phage-enzyme 100uJ of 50 fold concentrate of phage (repre-
senting 5mls of culture supernatant) was centrifuged through ultrafiltration membranes with nominal molecular weight
10 retention of 300,000 daltons. Western blots of flow through and retentate fractions were detected with anti-alkaline
phosphatase antiserum. The equivalent of 800uJ of original culture supernatant was run on the gel.
A. Phage were grown in TG1 cells, a) fd-phoAla166 before ultrafiltration (short exposure), b) fd-phoAla166 before
ultrafiltration, c) fd-phoAla166 material retained on ultrafiltration membrane.
15 b. Phage were grown in KS272 cells, a) fd-phoAla166 before ultrafiltration, b) fd-phoAla166 material retained on
ultrafiltration membrane, c) fdCAT2. d) fdCAT2 mixed with purified alkaline phosphatase before ultrafiltration, e)
Retentate from sample d. f) Flow through from sample d.
Figure 36 Electrophoresis of samples from stages of a Fab assembly. Samples from different stages in the PCR
20 Fab assembly process described in example 33 were subjected to electrophoresis on a 1 % TAE-agarose gel. Samples
from a comparable scFv assembly process (as in example 14) are shown for comparison. Samples left to right are:
M = Markers
VHCH1 = sequences encoding VHCH1 domains amplified by PCR
25 VKCK = sequences encoding VKCK domains amplified by PCR
-L = Fab assembly reaction performed in absence of linker
+L = Fab PCR assembly reaction product VHCH1 plus VKCK plus linker
M = Markers
VK = sequences encoding VK domain amplified by PCR
30 VL = sequences encoding VH domains amplified by PCR
-L = scFv assembly reaction in absence of linker
+L = scFv assembly reaction in presence of linker
M = Markers
35 Figure 37. Comparison of ELISA signals with scFv D1.3 cloned in fd-CAT2 (fd) or pCAT-3. pCAT-3 scFc1.3 has
been rescued with M13K07 (K07). M13K07Aglll No 3 (gill No 3) or M13K07 glllANo2 (g111No2). Phage antibodies
are compared at 10 times (10x) 1 times (1x) or 0.1 times (0.1x) concentrations relative to concentration in the super-
natant after overnight growth. The fdCAT2 and pCAT-3 non-recombinant vector signals were <0.01 at 10x concentra-
tion. M1 3K07 glllANo 1 did not rescue at all, as judged by no signal above background in this ELISA.
40 Figure 38. Western blot of PEG precipitated phage used in ELISA probed with anti-g3p. Free g3p and the g3p-
scFvDI .3 fusion bands are arrowed.
Sample 1 - fd scFcD1.3
Sample 2 - pCAT3 vector
45 Sample 3 - pCAT3 scFcD1.3 rescued with M13K07, no IPTG
Sample 4 - pCAT3 scFvD1.3 rescued with M13K07, 50uJv1 IPTG
Sample 5 - pCAT3 scFcD1.3 rescued with M13K07, 100uJv1 IPTG
Sample 6 - pCAT3 scFvD1.3 rescued with M13K07 glllANo3 (no IPTG)
Sample 7 - pCAT3 scFcD1.3 rescued with M13K07 glllA No 2 (no IPTG)
50
Panel A samples contain the equivalent of SjlxI of phagemid culture supernatant per track, and 80uJ of the fd su-
pernatant (10-fold lower phage yield than the phagemid). Panel B phagemid samples are those used in panel A at a
five-fold higher sample loading (equivalent to 40uJ of culture supernatant per track) to enable visualisation of the fusion
band in samples rescued with parental M13K07.
55 Figure 39 is a graph showing fdCAT2scFvD1 .3 enrichment produced from a mixture of fdCAT2scFvD1 .3 and
fdCAT2TPB4 by one round of panning.
Figure 40 is a graph showing fdCAT2scFvD1 .3 enrichment produced from a mixture of fdCAT2scFvD1 .3 and
fdCAT2TPB1 by one round of panning.
21
EP0 774 511 A1
Figure 41 . Western blot of phage proteins of fdCAT2(1 ) and fd-tet-SNase(2) with anti-g3p antiserum. Marker mo-
lecular weights bands are indicated(kD).
Figure 42. Nuclease assay of soluble SNase (3 ng)(A1), fd-tet-SNase(4 x 10 9 TU,(B-1 ),fd-CAT2(2 x 10 10 TU)(C-1)
and of a PEG-precipitated fdCAT2 and SNase mixture(2 x 10 10 TU and 0.7ug)(D-1) in a 10-fold dilution series (1 to 3
5 or 4). Marker (M) is a Hindi 1 1 digest of VDNA(New England Biolabs).
Figure 43. ELISA signals obtained with fd-tet, fd-CD4-V1 and fd-CD4-V1 V2. In each group of three, the samples
are left to right phage concentrate(SN); phage concentrate plus soluble CD4(SN + sCD4); phage concentrate plus gp
120 (SN + gp 120).
Figure 44. shows the DNA sequence of scFv B1 8 (anti-NP).
10 Figure 45 shows a map of the insert of sequences encoding FvD1.3 present in fd-tet FvD1.3 (example 39). rbs
designates the ribosome binding site. Gene III is now shown in its full length.
Figure 46. shows an ELISA assay of phages displaying FvD1.3 or scFvD1.3 by binding to plates coated with
lysogyme. Signals obtained at various dilution factors are shown. FvD1 .3 (AS-Stuffer) which does not express Fv was
used as a control.
15 Figure 47. shows a schematic representation of steps involved in the PCR assembly of nucleotide sequences
encoding human Fab fragments. Details are in example 40.
Figure 48. shows A. a map of plasmid pJM1-FabD1.3 which is used for the expression of soluble human Fab
fragments and as a template for the synthesis of linker DNA for Fab assembly. B. a schematic representation of se-
quences encoding a Fab construct. C. The sequence of DNA template for the synthesis of linker DNA for Fab assembly.
20 Figure 49. shows a schmatic representation of steps involved in the PCR assembly of nucleotide sequences en-
coding human scFv fragments. Details are in example 42.
Figure 50. ELISA assay of phage antibodies using plates coated with turkey egg lysogyme. Two clones B1 and
A4 are shown derived by mutagenesis and selection from pAbD1 .3 (example 45). Concentration ( x axis) refers to the
concentration of phage for each sample relative to the concentration in culture supernatant. B1 has raised binding to
25 turkey egg lysogyme compared to D1 .3. A4 has reduced binding to hen egg lysogyme compared to D1 .3.
Figure 51 . ELISA of phage antibodies binding to HEL and TEL. Clone 1 is fdCAT2scFvD1 .3. Clones 2 to 1 0 were
obtained from the library (example 46) after selection. The background values as defined by binding of these clones
to BSA were subtracted.
Figure 52. shows the DNA sequence of the light chains D1 .3 M1 F and M21 derived by selection from a hierarchical
30 library in example 46.
Figure 53 shows a Fv lambda expression vector (example 48) derived from pUC119. It contains the rearranged
lambdal germ line gene. The heavy and light chain cassettes each contain a ribosome binding site upstream of the pel
B leader (Restriction sites shown as: H=Hind III; Sp=Sphl; B=BamHI, E=EcoRI.
35 Materials and Methods
The following procedures used by the present applicants are described in Sambrook, J. at al., 1989 supra.: re-
striction digestion, ligation, preparation of competent cells ( Hanahan method ), transformation, analysis of restriction
enzyme digestion products on agarose gels, purification of DNA using phenol/chloroform, 5'-end labelling of oligonu-
40 cleotides, filter screening of bacterial colonies, preparation of 2xTY medium and plates, preparation of tetracycline
stock solutions, PAGE of proteins, preparation of phosphate buffered saline.
All enzymes were supplied by New England Biolabs (CP Laboratories, PO Box 22, Bishop's Stortford, Herts.,
England) and were used according to manufacturer's instructions unless otherwise stated.
The vector fd-tet (Zacher, A.N. atal., 1980, supra) was obtained from the American Type Culture Collection (ATCC
45 No. 37000) and transformed into competent TG 1 cells (genotype: K1 28 (lac-pro), sup E, thi, hsdD5/F traD36, pro A+B+,
Lac |q, lac 5M15).
Viral particles were prepared by growing TG1 cells containing the desired construct in 10 to 100 mis 2xTY medium
with 1 5 |ug/ml tetracycline for 1 6-24 hours. The culture supernatant was collected by centrif ugation for 1 0 mins at 1 0,000
rpm in an 8 x 50 ml rotor, Sorval RC-5B centrifuge. Phage particles were precipitated by adding 1/5th volume 20%
so polyethylene glycol (PEG)/2.5M NaCI and leaving at 4°C for 1 hour. These were spun for 15 minutes as described
above and the pellets resuspended in 10 mM Tris/HCI pH 8, 1mm EDTAto 1/1 00th of the original volume. Residual
bacteria and undissolved material were removed by spinning for 2 minutes in a microcentrifuge. Single stranded DNA
for mutagenesis or sequencing was prepared from concentrated phage according to Sambrook, J., at al., 1 989,, supra.
55
22
EP0 774 511 A1
Index of Examples
Example 1 Design of Insertion Point Linkers and Construction of Vectors
5 This example covers the construction of two derivatives of the phage vector fd-tet: a) fdTPs/Bs for the insertion of
VH coding sequences; and b) fdTPs/Xh for the insertion of scFv coding sequences. The derivative vectors have a new
BstEII site for insertion of sequences.
Example 2 Insertion of Immunoglobulin Fv Domain into Phage
10
This example covers the insertion of scFv coding sequences derived from an anti-lysozyme antibody D1.3 into
fdTPs/Xh to give the construct fdTscFvDI .3.
Example 3 Insertion of Immunoglobulin VH Domain into Phage
15
This example covers the insertion of VH coding sequences derived from an anti-lysozyme antibody D1.3 into
fdTPs/Bs to give the construct fdTVHDI .3.
Example 4 Analysis of Binding Specificity of Phage Antibodies
20
This example investigates the binding specificities of the constructs fdTscFcDI .3 and fdTVHDI .3.
Example 5 Construction of fdCAT2
25 This example covers the construction of the derivative fdCAT2 of the phage vector fdTPs/Xh. The derivative has
restriction sites for enzymes that cut DNA infrequently
Example 6 Specific Binding of Phage Antibody (pAb) to Antigen
30 This example shows the binding of pAb fdTscFvDI .3 to lysozyme by ELISA.
Example 7 Expression of FabD1 .3
This example concerns the display of an antibody Fab fragment at the phage surface. The VH-CH1 chain is ex-
35 pressed by fdCAT2. The VL-CL chain is expressed by pUC1 9 in a bacterial host cell also infected with fdCAT2.
Example 8 Isolation of Specific, Desired Phage from a Mixture of Vector Phage
This example shows how a phage (e.g. fdTscFcD1.3) displaying a binding molecule can be isolated from vector
40 phage by affinity techniques.
Example 9 Construction of pAb Expressing Anti-Hapten Activity
This example concerns the insertion of scFv coding sequences derived from the anti-oxazolone antibody NQ11
45 into fdTPs/Xh to generate the construct pAbNQ1 1 . The example shows the binding of pAbNQ1 1 to oxazalone by ELISA.
Example 10 Enrichment of pAbD1 .3 from Mixtures of other pAbs by Affinity Purification
This example shows how a phage (eg. pAbD1 .3) displaying one sort of biding molecule can be isolated from phage
50 (e.g. pAbNQ1 1 ) displaying another sort of binding molecule by affinity techniques.
Example 11 Insertion of a Gene Encoding an Enzyme (Alkaline Phosphate) into fdCAT2
This example concerns the invention of coding sequences for an enzyme into the vector fdCAT2 to give the phage
55 enzyme, fdphoAlal 16.
23
EP0 774 511 A1
Example 12 Measuring Enzyme Activity Phage - Enzyme
This example shows the functionality of an enzyme (alkaline phosphatase) when displayed at the phage surface
(fdphoAla166).
5
Example 13 Insertion of Binding Molecules into Alternative Sites in the Phage
This example covers the insertion of scFv coding sequences derived from a) the anti-lysozyme antibody D1 .3; and
b) the anti-oxazalone antibody NQ11 into a BamHI site of fdTPs/Xh to give the constructs fdTBaml having an NQ11
10 insert.
Example 14 PCR Assembly of Mouse VH and VLK Repertoires for Phage Display
This example concerns a system for the display on phage of all VH and VLK repertoires encoded by a mouse.
15 The system involves the following steps. 1 ) Preparation of RNA from spleen. 2) Preparation of cDNA from the RNA 3)
Use of primers specific for antibody sequences to PCR amplify all VH and VLK cDNA coding sequences 4) Use of
PCR to create a linker molecule from linking pairs of VH and VLK sequences 5) Use of PCR to assemble continuous
DNA molecules each comprising a VH sequence, a linker and a VLK sequence. The specific VHA/LK combination is
randomly derived 6) Use of PCR to introduce restriction sites.
20
Example 15. Insertion of the Extracellular Domain of a Human Receptor for Platelet Derived Growth Factor (PDGF)
Isoform BB into fdCAT2
This example concerns the insertion of coding sequences for the extracellular domain of the human receptor for
25 PDGF into the vector fdCAT2 to give the construct fdhPDGFBR.
Example 16. Binding of 125 l-PDGF-BB to the Extracellular Domain of the Human Receptor for PDGF Isoform BB
Displayed on the Surface of fd Phage. Measured using an Immunoprecipitation Assay .
30 This example shows that the human receptor PDGF Isoform BB is displayed on the surface of the phage in a form
which has the ability to bind its ligand.
Example 17. Construction of Phagemid Containing Gene III Fused with the Coding Sequence for a Binding Molecule .
35 This example concerns the construction of two phagemids based on pUC119 which separately contain gene III
from fdCAT2 and the gene III scFv fusion fdCAT2seFvD1 .3 to generate pCAT2 and pCAT3 scFvDI .3 respectively.
Example 18. Rescue of Anti-Lvsozyme Antibody Specificity from pCAT3scFvD1 .3 by M13KQ7
40 This example describes the rescue of the coding sequence for the gene lllscFv fusion from pCAT3scFvD1 .3 by
M13M07 helper phage growth, phage were shown to be displaying scFv anti-lypozyme activity by ELISA.
Example 19. Transformation Efficiency of PCAT-3 and pCAT-3 scFvDI .3 Phagemids
45 This example compared the efficiency of the phagemids pVC119, pCAT-3 and pCAT3scFvD1 .3 and the phage
fdCAT2 to transform E.coli.
Example 20 PCR Assembly of a Single Chain Fv Library from an Immunised Mouse
50 This example concerns a system for the display on phage of scFv (comprising VH and VL) from an immunised
mouse using the basic technique outlined in example 1 4 (cDNA preparation and PCR assembly of the mouse VH and
VLK repertoires) and ligating the PCR assembled sequences into fdCAT2 to create a phage library of 10 5 -clones.
Testing of 500 clones showed that none showed specificity against phox.
55 Example 21 . Selection of Antibodies Specific for 2-phenyl-5-oxazolone from a Repertoire from an Immunised Mouse.
This example shows that phage grown from the library established in example 20 can be subjected to affinity
selection using phOXto select those phage displaying scFv with the desired specificity.
24
EP0 774 511 A1
Example 22. Generation of Further Antibody Specificities by the Assembly of Hierarchial Libraries.
This example concerns the construction of hierarchial libraries in which a given VH sequence is combined with
the complete VLK repertoire and a given VLK sequence is combined with the complete VH repertoire and selection
s from these libraries of novel VH and VL pairings.
Example 23. Selection of Antibodies Displayed on Bacteriophage with Different Affinities for 2-phenyl-5-oxazolone
using Affinity Chromatography
10 This example concerns the separation by affinity techniques of phages displaying scFv fragments with differing
binding affinities for a given antigen.
Example 24. Construction of Phagemid pHEN1 for the Expression of Antibody Fragments Expressed on the surface
of Bacteriophage following Superinfection
15
This example concerns the construction of the phagemid pHEN1 derived from pUC11 9. pHEN1 has the features
shown in Fig. 26.
Example 25. Display of Single Chain Fv and Fab Fragments Derived from the Anti-Oxazolone Antibody NQ 10.12.5
20 on Bacteriophage fd using pHEN1 and fdCAT2.
This example describes the display of scFv and Fab fragment with a specificity against phOx on the surface of a
bacteriophage. For display of scFv the phagemid pHEN1 comprises the sequences encoding scFv (VH and VL) for
rescue by either the phages VSM13 or fdCAT2. For display of Fab the phage fdCAT2 comprises the sequence for
25 either the H or L chain as a fusion with g3p and the phagemid pHEN1 comprises the sequence for the appropriate H
or L chain partner.
Example 26. Rescue of Phagemid Encoding a Gene III protein Fusion with Antibody Heavy or Light Chains by Phage
Encoding the Complementary Antibody Displayed on Phage and the Use of this Technique to make Dual Combinatorial
30 Libraries
This example covers the use of phage antibodies encoding the antibody heavy or light chain to rescue a phagemid
encoding a gene 3 protein fusion with the complementary chain and the assay of Fab fragments displayed on phage
in ELISA. The use of this technique in the preparation of a dual combinatorial library is discussed.
35
Example 27 Induction of Soluble scFv and Fab Fragments using Phagemid pHEN1
This example covers the generation of soluble scFv and Fab fragments from gene III fusions with sequences
encoding these fragments by expression of clones in pHEN1 in an E.coli strain which does not suppress amber mu-
40 tations.
Example 28 Increased Sensitivity in ELISA of Lysozyme using fdTscFvDI .3 as Primary Antibody compared to Soluble
scFvD1.3
45 This example covers the use of fdTscFvDI .3 in ELISA showing that lower amounts of lysozyme can be detected
with phage antibody fdTscFvDI .3 than with soluble scFvDI .3.
Example 29 Direct Rescue and Expression of Mouse Monoclonal Antibodies as Single Chain Fv Fragments on the
Surface of Bacteriophage fd
50
This example covers the display on phage as functional scFv fragments of two clones directly derived from cells
expressing monoclonal antibodies directed against oestriol. Both clones were established to be functional using ELISA.
Example 30 Kinetic Properties of Alkaline Phosphatase Displayed on the Surface of Bacteriophage fd
55
This example concerns the demonstration that the kinetic properties of an enzyme, alkaline phosphatase, displayed
on phage are qualitatively similar to those of the same enzyme when in solution.
25
EP0 774 511 A1
Example 31 Demonstration using Ultrafiltration that Cloned Alkaline Phosphatase Behaves as Part of the Virus Particle
This example concerns the construction of the phage enzyme fdphoArg166 and the demonstration that both the
fusion protein made and the catalytic activity observed derive from the phage particle.
5
Example 32 Affinity Chromatography of Phage Alkaline Phosphatase
This example concerns the binding of alkaline phosphatase displayed on phage to an arsenate-Sepharose affinity
column and specific elution of these phage using the reaction product, phosphate.
w
Example 33 PCR Assembly of DNA Encoding the Fab Fragment of an Antibody Directed against Oxazolone
This example covers the construction of a DNA insert encoding a Fab fragment by separate amplification of heavy
and light chain DNA sequences followed by assembly. The construct was then inserted into the phage vector fdCAT2
15 and the phagemid vector pHEN1 and the Fab fragment displayed on the surface was shown to be functional.
Example 34 Construction of a Gene III Deficient Helper Phage
This example describes the construction of a helper phage derived from M1 3K07 by deleting sequences in gene
20 in. Rescue of pCAT3-scFvD1 .3 is described. The scFvDI .3 is expressed at a high level as a fusion using the deletion
phage, equivalent to expression using fdCAT2-scFvD1 .3.
Example 35 Selection of bacteriophage expressing scFv fragments directed against lysozyme from mixtures according
to affinity using a panning procedure
25
This example concerns the selection of bacteriophage according to the affinity of the scFvf ragment directed against
lysozyme which is expressed on their surface. The phage of different affinities were bound to Petri dishes coated with
lysozyme and, following washing, bound phage eluted using triethylamine. Conditions were found where substantial
enrichment could be obtained for a phage with a 5-fold higher affinity than the phage with which it was mixed.
30
Example 36 Expression of Catalvtically Active Staphylococcal Nuclease on the Surface of Bacteriophage fd
This example concerns the construction of a phage enzyme which expresses Staphylococcal nuclease and the
demonstration that the phage enzyme retains nuclease activity.
35
Example 37 Display of the Two Aminoterminal Domains of Human CD4 on the Surface of fd Phage
This example covers the cloning of genes for domains of CD4, a cell surface receptor and member of the immu-
noglobulin superfamily, into bacteriophage fd. The receptor is shown to be functional on the surface of phage by binding
40 to the HIV protein gp1 20.
Example 38 Generation and Selection of Mutants of an Anti-4-hydroxy-3-nitrophenylacetic acid (NP) Antibody
expressed on Phage using Mutator strains
45 This example covers the introduction of mutations into a gene for an antibody cloned in phage by growth of the
phage in strains which randomly mutate DNA due to defects in DNA replication. Several mutations are introduced into
phage which can then be selected from parent phage.
Example 39 Expression of a Fv Fragment on the Surface of Bacteriophage by Non-Covalent Association of VH and
so VL domains
This example shows that functional Fv fragments can be expressed on the surface of bacteriophage by non-
covalent association of VH and VL domains. The VH domain is expressed as a gene III fusion and the VL domain as
a soluble polypeptide. Sequences allowing expression of these domains from the anti-lysozyme antibody D1 .3 in this
55 form were introduced into phage and the resulting displayed Fv fragment shown to be functional by ELISA.
26
EP0 774 511 A1
Example 40 A PCR Based Technique for one step Cloning of Human V-genes as Fab Constructs
This example gives methods for the assembly of Fab fragments from genes for antibodies. Examples are given
for genes for antibodies directed against Rhesus-D in a human hybridoma and a polyclonal lymphoblastic cell line.
5
Example 41 Selection of Phage Displaying a Human Fab Fragment directed against the Rhesus-D Antigen by binding
to Cells displaying the Rhesus D Antigen on their Surface
This example concerns the construction of, and display of phage antibodies from, a phagemid encoding a human
10 Fab fragment directed against the Rhesus D antigen. Phage displaying this antigen were then affinity selected from a
background of phage displaying scFvDI .3 anti-lysozyme on the basis of binding to Rhesus-D positive red blood cells.
Example 42 A PCR Based Technique for One Step Cloning of Human scFv Constructs
15 This example describes the generation of libraries of scFv fragments derived from an unimmunized human. Ex-
amples are given of the preparation for phage display of libraries in phagemids of scFv fragments derived from IgG
and IgM sequences.
Example 43 Isolation of Binding Activities from a Library of scFvs from an Unimmunized Human
20
This example describes the isolation, from the library of scFv fragments derived from IgM genes of an unimmunized
human, of clones for phage antibodies directed against BSA, lysozyme and oxazolone. Selection was by panning or
affinity chromatography and analysis of binding specificity by ELISA. Sequencing of the clones showed them to be of
human origin.
25
Example 44 Rescue of human IgM library using helper phage lacking gene 3 ( g3)
This example covers the isolation, from the library of scFv fragments of unimmunized human IgM genes, of clones
of phage antibodies of clones for phage antibodies specific for thyroglobulin and oxazolone. In this example rescue
30 was with M13K07glll No3 (NCTC12478), a helper phage defective in gene III. Fewer rounds of selection appeared
necessary for a phagemid library rescued with this phage compared to one rescued with M13K07.
Example 45 Alteration of Fine Specificity of scFvDI .3 displayed on Phage by Mutagenesis and Selection on Immobilized
Turkey Lysozyme
35
This example covers the in vitro mutagenesis of pCATscFcDI .3 by replacement, with random amino acids, of
residues known to be of importance in the preferential recognition of hen egg lysozyme over turkey egg lysozyme by
scFvDI .3. Following selection for phage antibodies recognising turkey egg lysozyme by affinity chromatography, clones
were analysed for specificity by ELISA. Two groups of clones were found with more equal recognition of hen and turkey
40 lysozymes, one with increased ELISA signal with the turkey enzyme and one with reduced signal for the hen enzyme.
Example 46 Modification of the Specificity of an Antibody by Replacement of the VLK Domain by a VLK Library derived
from an Unimmunised Mouse
45 This example shows that replacement of the VL domain of scFvD1.3 specific for hen eggwhite lysozyme (HEL)
with a library of VL domains allows selection of scFv fragments which bind also to turkey eggwhite lysozyme (TEL).
The scFv fragments were displayed on phage and selection by panning on tubes coated with TEL. Analysis by ELISA
showed clones with enhanced binding to TEL compared to HEL. Those with highest binding to TEL were sequenced.
so Example 47 Selection of a Phage Antibody Specificity by binding to an Antigen attached to Magnetic Beads. Use of a
Cleavable Reagent to allow Elution of Bound Phage under Mild Conditions
This examples covers the use of a cleavable bond in the affinity selection method to alow release of bound phage
under mild conditions. pAbNQ11 was enriched approximately 600 fold from a mixture with pAbD1 .3 by selection using
55 biotinylated Ox-BSA bound to magnetic beads. The cleavage of a bond between BSA and the biotin allows elution of
the phage.
27
EP0 774 511 A1
Example 48 Use of Cell Selection to provide an Enriched Pool of Antigen Specific Antibody Genes, Application to
reducing the Complexity of Repertoires of Antibody Fragments Displayed on the Surface of Bacteriophage
This example covers the use of cell selection to produce an enriched pool of genes encoding antibodies directed
5 against 4-hydroxy-3-nitrophenylacetic acid and describes how this technique could be used to reduce the complexity
of antibody repertoires displayed on the surface of bacteriophage.
Example 1
10 Design of Insertion Point Linkers and Construction of Vectors
The vector fd-tet has two BstEII restriction sites flanking the tetracycline resistance gene (fig 3). Since the strategy
for inserting the VH fragments was to ligate them into a newly inserted BstEII site within gene III, it was advantageous
to delete the original BstEII sites from fd-tet. This was achieved by digesting fd-tet with the restriction enzyme BstEII,
15 filling-in the 5' overhangs and re-ligating to generate the vector fdT8Bst. Digestion of fd-tet with BstEII (0.5 units/uJ)
was carried out in 1x KGB buffer (100 mM potassium glutamate, 23 mM Tris-acetate (pH 7.5), 10 mM magnesium
acetate, 50 ujg/ml bovine serum albumin, 0.5 mM dithiothreitol (Sambrook, J., at al., 1989, supra.) with DNA at a
concentration of 25 ng/ul The 5' overhang was filled in, using 2x KGB buffer, 250 jlxM each dNTP's (Pharmacia Ltd.,
Pharmacia House, Midsummer Boulevard, Milton Keynes, Bucks., UK. ) and Klenow Fragment (Amersham Interna-
20 tional, Lincoln Place, Green End, Aylesbury, Bucks., UK) at 0.04 units/ul After incubating for 1 hour at room temper-
ature, DNA was extracted with phenol/chloroform and precipitated with ethanol.
Ligations were carried out at a DNA concentration of 50ng/uJ). Ligations were transformed into competent TG1
cells and plated onto TY plates supplemented with 15 pg/ml tetracycline. This selects for vectors where the gene for
tetracycline resistance protein has reinserted into the vector during the ligation step. Colonies were picked into 25 mis
25 of 2xTY medium supplemented with 15 u.g/ml tetracycline and grown overnight at 37°C.
Double stranded DNA was purified form the resulting clones using the gene-clean II kit (Bio101 Inc., PO Box 2284,
La Jolla, California, 92038-2284, USA.) and according to the small scale rapid plasmid DNA isolation procedure de-
scribed therein. The orientation of 5 of the resulting clones was checked using the restriction enzyme Clal. A clone
was chosen which gave the same pattern of restriction by Clal as fd-tet, but which had no BstE II sites.
30 in vitro mutagenesis of fdT6Bst was used to generate vectors having appropriate restriction sites that facilitate
cloning of antibody fragments downstream of the gene III signal peptide and in frame with the gene III coding sequence.
The oligonucleotide directed mutagenesis system version 2 (Amersham International) was used with oligo 1 (figure 4)
to create fdTPs/Bs (to facilitate cloning of VH fragments). The sequence offdTPs/Bs (figure 4) was confirmed using
the sequenase version 2.0 kit (USB Corp., PO Box 22400, Cleveland, Ohio, 44122, UsA.) with oligo 3 (figure 4) as a
35 primer.
A second vector fdTPs/Xh (to facilitate cloning of single chain Fv fragments) was generated by mutagenising fdTPs/
Bs with oligo 2 according to the method of Venkitaraman, A.R., Nucl. Acid Res. J_7, p 3314. The sequence of fdTPs/
Xh (figure 4) was confirmed using the sequenase version 2.0 kit (USB Corp.) with oligo 3 as a primer.
Clearly, alternative constructions will be apparent to those skilled in the art. For example, M13 and/or its host
40 bacteria could be modified such that its gene III could be disrupted without the onset of excessive cell death; the
modified fd gene III, or other modified protein, could be incorporated into a plasmid containing a single stranded phage
replication origin, such as pUC119, superinfection with modified phage such as K07 would then result in the encapsu-
lation of the phage antibody genome in a coat partially derived from the helper phage and partly from the phage antibody
gene III construct.
45 The detailed construction of a vector such as fdTPs/Bs is only one way of achieving the end of a phage antibody.
For example, techniques such as sticky feet cloning/mutagenesis (Clackson, T. and Winter, G. 1 989 Nucl. Acids. Res.,
17 , p 10163-10170) could be used to avoid use of restriction enzyme digests and/or ligation steps.
Example 2.
50
Insertion of Immunoglobulin Fv Domain into Phage
The plasmid scFv D1 .3 myc (gift from g. Winter and A. Griffiths) contains VH and VL sequences from the antibody
D1 .3 fused via a peptide linker sequence to form a single chain Fv version of antibody D1 .3. The sequence of the scFv
55 and surrounding sequences in scFcDI .3 myc is shown in figure 5.
The D1.3 antibody is directed against hen egg lysozyme (Harper, M. at al., 1987, Molec. Immunol. 24, 97-108)
and the scFv form expressed in E.coli has the same specificity (A. Griffiths and G. Winter personal Communication).
Digestion of scFv D1.3 myc with Pst1 and Xhol (these restriction sites are shown on Fig. 5), excises a fragment
28
EP0 774 511 A1
of 693 bp which encodes the bulk of the scFv. Ligation of this fragment into fdTPs/Xh cleaved with Pst1 and Xhol gave
rise to the construct fdTscFcDI .3 encoding the gene III signal peptide and first amino acid fused to the complete D1 .3
scFv, followed by the mature gene III protein from amino acid 2.
The vector fdTPs/Xh was prepared for ligation by digesting with the Pst1 and Xhol for 2 hours followed by digestion
5 with calf intestinal alkaline phosphatase (Boehringer Mannheim UK Ltd., Bell Lane, Lewes, East Sussex, BN7 1LG)
at one unit/ul for 30 minutes at 37°C. Fresh calf intestinal alkaline phosphatase was added to a final total concentration
of 2 units/ul and incubated for a further 30 minutes at 37°C. The reaction was extracted three times with phenol/
chloroform, precipitated with ethanol and dissolved in water. The insert from scFcDI .3 myc was excised with the ap-
propriate restriction enzymes (Pstl and Xhol) extracted twice with phenol/chloroform, precipitated with ethanol and
10 dissolved in water. Ligations were carried out as described in example 1 , except both vector and insert samples were
at a final concentration of 5 ng/ul each. The formation of the correct construct was confirmed by sequencing as described
in example 1 .
To demonstrate that proteins of the expected size were produced, virions were concentrated by PEG precipitation
as described above. The samples were prepared for electrophoresis as described in Sambrook J. at al 1989 supra.
15 The equivalent of 2mls of supernatant was loaded onto an 1 8% SDS polyacrylamide gel. After electrophoresis, the gel
was soaked in gel running buffer (50 mM tris, 380 mM Glycine, 0.1%SDS) with 20% methanol for 15 minutes. Transfer
to nitrocellulose filter was executed in fresh 1x running buffer/20% methanol using TE70 Semi Phor a semi-dry blotting
apparatus (Hoeffer, 654 Minnesota Street, Box 77387, San Francisco, California 94107, USA.).
AFter transfer, the filter was blocked by incubation for 1 hour in a 2% solution of milk powder (Marvel) in phosphate
20 buffered saline (PBS). Detection of scFv and VH protein sequences in the phage antibody fusion proteins was effected
by soaking the filter for 1 hour with a 1 h 000 dilution (in 2% milk powder) of a rabbit polyclonal antiserum raised against
affinity purified, bacterially expressed scFv fragment (gift from G. Winter). After washing with PBS (3x5 minute washes),
bound primary antibody was detected using an anti-rabbit antibody conjugated to horseradish peroxidase (Sigma,
Fancy Road, Poole, Dorset, BH17 7NH, UK.) for 1 hour. The filter was washed in PBS/0. 1 % triton X-1 00 and developed
25 with 0.5 mg/ml 3,3'-diaminobenzidine tetrahydrochloride (DAB), 0.02% cobalt chloride, 0.03% hydrogen peroxide in
PBS.
The results showed that with clones fdTVHD1.3 (from example 3 incorporating sequences coding for VH) and
fdTscFvD1.3 (incorporating sequences coding for scFv) a protein of between 69,000 and 92,500 daltons is detected
by the anti-Fv serum. This is the expected size for the fusion proteins constructed. This product is not observed in
30 supernatants derived from fd-tet, fdT5Bst or fdTPs/Xh.
Example 3.
Insertion of Immunoglobulin VH Domain into Phage Antibody
35
The VH fragment from D1. 3 was generated from the plasmid pSW1 -VHD1 .3-TAG1 (Ward, E.S.atal., 1989 supra.).
Digestion of this plasmid with Pst1 and BstEII generates the fragment shown between positions 113 and 432 in figure
5. Cloning of this fragment into the Pst1 and BstEII sites of fdTPs/Bs gave rise to the construct fdTVHD1.3 which
encodes a fusion protein with a complete VH domain inserted between the first and third amino acids of the mature
40 gene III protein (amino acid two has been deleted).
The methods used were exactly as in example 2 except that the vector used was fdTPs/Bs digested with Pst1 and
BstEII.
Example 4.
45
Analysis of Binding Specificity of Phage Antibodies
The binding of the various phage antibodies to the specific antigen, lysozyme, was analysed using ELISA tech-
niques. Phage antibodies (e.g. fdTVHDI .3 and fdTsc/FvD1 .3) were grown in E.coli and Phage antibody particles were
so precipitated with PEG as described in the materials and methods. Bound phage antibody particles were detected using
polyclonal sheep serum raised against the closely related phage M13.
ELISA plates were prepared by coating 96 well plates (Falcon Microtest III flexible plate. Falcon: Becton Dickinson
Labware, 1950 Williams Drive, Oxnard, California, 93030, USA.) with 200 ul of a solution of lysozyme (1 mg/ml unless
otherwise stated) in 50 mm NaHC03 for 16-24 hours. Before use, this solution was removed, the plate rinsed several
55 times in PBS and incubated with 200 ul of 2% milk powder/PBS for 1 hour. AFter rinsing several times with PBS, 100
ul of the test samples were added and incubated for 1 hour. Plates were washed (3 rinses in 0.05% Tween 20/PBS
followed by 3 rinses in PBS alone). Bound phage antibodies were detected by adding 200 ul/well of a 1/1000 dilution
of sheep anti-M13 polyclonal antiserum (gift from G. Winter, although an equivalent antibody can be readily made by
29
EP0 774 511 A1
one skilled in the art using standard methodologies) in 2% milk powder/PBS and incubating for 1 hour. After washing
as above, plates were incubated with biotinylated anti-sheep antibody (Amersham International) for 30 minutes. Plates
were washed as above, and incubated with streptavidinhorseradish peroxidase complex (Amersham International).
After a final wash as above, 0.5 mg/ml ABTS substrate in citrate buffer was added (ABTS = 2'2'-azinobis (3-ethylben-
5 zthiazoline sulphonic acid); citrate buffer = 50 mM citric acid, 50 mM tri-sodium citrate at a ratio of 54:46. Hydrogen
peroxide was added to a final concentration of 0.003% and the plates incubated for 1 hour. The optical density at 405
nm was read in a Titertek multiskan plate reader.
Figure 6 shows the effect of varying the amount of phage antibody. 100 ul of various dilutions of PEG precipitated
phage were applied and the amount expressed in terms of the original culture volume from which it was derived. Signals
10 derived from both the scFv containing phage antibody (fdTscFvDI .3) and the VH containing phage antibody
(fdTVHD1.3) and the VH containing phage antibody were higher than that derived from the phage antibody vector
(fdTPs/Xh). The highest signal to noise ratio occurs using the equivalent of 1 .3 mis of culture.
Figure 7 shows the results of coating the plates with varying concentrations of lysozyme or bovine serum albumin
(BSA). The equivalent of 1 ml of the original phage antibody culture supernatant was used. The signals from super-
15 natants derived from fdTscFvDI .3. were again higher than those derived from fdTPs/Xh when lysozyme coated wells
were used. There was no significant difference between these two types of supernatant when the plates were coated
with BSA. Broadly speaking the level of signal on the plates is proportional to the amount of lysozyme coated. These
results demonstrate that the binding detected is specific for lysozyme as the antigen.
20 Example 5.
Construction of fd CAT 2
It would be useful to design vectors that enable the use of restriction enzymes that cut DNA infrequently thus
25 avoiding unwanted digestion of the antibody gene inserts within their coding sequence. Enzymes with an eight base
recognition sequence are particularly useful in this respect, for example Not1 and Sfil. Chaudhary at al (PNAS 87
p1 066-1 070, 1990) have identified a number of restriction sites which occur rarely in antibody variable genes. The
applicant has designed and constructed a vector that utilises two of these sites, as an example of how this type of
enzyme can be used. Essentially sites for the enzymes Apal_1 and Not1 were engineered into fdTPs/Xh to create
30 fdCAT2.
The oligonucleotide:
5' ACT TTC AAC AGT TTC TGC GGC CGC CCG TTT GAT CTC GAG CTC
35 CTG CAG TTG GAC CTG TGC ACT GTG AGA ATA GAA 3'
was synthesised (supra fig 4 legend) and used to mutagenise fdTPs/Xh using an in vitro mutagenesis kit from Amer-
sham International as described in example 1 , to create fd-CAT2. The sequence of fd-CAT2 was checked around the
site of manipulation by DNA sequencing. The final sequence around the insertion point within gene III is shown in figure
40 8.
N.B. fdCAT2 is also referred to herein by the alternative terminologies fd-tet-DOG1 and fdDOGI .
Example 6
45 Specific Binding of Phage-antibody (pAb) to Antigen
The binding of pAb D1 .3 (fdTscFvDI .3 of example 2) to lysozyme was further analysed by ELISA.
Methods.
50
1 . Phage growth.
Cultures of phage transduced bacteria were prepared in 10-100 mis 2 x TY medium with 15uxj/ml tetracycline and
grown with shaking at 37°C for 16-24 hrs. Phage supernatant was prepared by centrifugation of the culture (10 min at
55 10,000 rpm, 8 x 50 ml rotor, Sorval RC-5B centrifuge). At this stage, the phage titre was 1 - 5 x 10 10 /ml transducing
units. The phage were precipitated by adding 1/5 volume 20% PEG 2.5 M NaCI, leaving for 1 hr at 4°C, and centrifuging
(supra). The phage pellets were resuspended in 10 mM Tris-HCI, 1 mM EDTA pH 8.0 to 1/1 00th of the original volume,
and residual bacteria and aggregated phage removed by centrifugation for 2 min in a bench microcentrifuge.
30
EP0 774 511 A1
ELISA
Plates were coated with antigen (1 mg/ml antigen) and blocked as described in example 4. 2 x 10 10 phage trans-
ducing units were added to the antigen coated plates in phosphate buffered saline (PBS) containing 2% skimmed milk
5 powder (MPBS). Plates were washed between each step with three rinses of 0.5% Tween-2G in PBS followed by three
rinses of PBS. Bound phage was developed by incubating with sheep anti-M1 3 antisera and detected with horseradish
peroxidase (HRP) conjugated anti-goat serum (Sigma, Poole, Dorset, UK) which also detects sheep immunoglobulins
and ABTS (2'2'-azinobis (3-ethylbenzthiazoline sulphonic acid). Readings were taken at 405 nm after a suitable period.
The results (figure 9) show that the antibody bearing-phage had the same pattern of reactivity as the original D1.3
10 antibody (Harper, M., Lema, R, Boulot, G., and Poljak, RJ. (1987) Molec. Immunol. 24, 97-108), and bound to hen
egg-white lysozyme, but not to turkey egg-white lysozyme, human lysozyme or bovine serum albumin. The specificity
of the phage is particularly illustrated by the lack of binding to the turkey egg-white lysozyme that differs from hen egg-
white lysozyme by only 7 amino acids.
15 Example 7.
Expression of Fab D1.3
The aim of this example was to demonstrate that the scFv format used in example 2 was only one way of displaying
20 antibody fragments in the pAb system. A more commonly used antibody fragment is the Fab fragment (figure 1) and
this example describes the construction of a pAb that expresses a Fab-like fragment on its surface and shows that it
binds specifically to its antigen. The applicant chose to express the heavy chain of the antibody fragment consisting
of the VH and CH1 domains from coding sequences within the pAb itself and to co-express the light chain in the bacterial
host cell infected with the pAb. The VH and CH1 regions of anti-lysozyme antibody D1 .3 were cloned in fd CAT2, and
25 the corresponding light chain cloned in plasmid pUC1 9. The work of Skerra and Pluckthun (Science 240, p1 038-1 040
(1988) and Better at al 1988 supra; demonstrated that multimeric antigen binding fragments of the antibody molecule
could be secreted into the periplasm of the bacterial cell in a functional form using suitable signal sequences. However,
in these publications, special measures were described as being needed to recover the binding protein from the cell,
for example Skerra and Pluckham needed to recover the Fv fragment from the periplasm by affinity chromatography.
30 The present applicants have shown that it is possible to direct the binding molecule to the outside of the cell on a phage
particle, a process that requires several events to occur: correct secretion and folding of the binding molecule; asso-
ciation of the chains of the binding molecule; correct assembly of the phage particle; and export of the intact phage
particle from the cell.
Alternatively, it is possible however, to express the light chain from within the pAb genome by, for example, cloning
35 an expression cassette into a suitable place in the phage genome. Such a suitable place would be the intergenic region
which houses the multicloning sites engineered into derivative of the related phage M13 (see, for example, Yanisch-
Perron, C. at al., Gene 33, p103-119, (1985)).
The starting point for this example was the clone Fab D1.3 in pUC19, a map of which is shown in figure 10. The
regions hybridising with the oligonucleotides KSJ6 and 7 below are shown underlined in fig 1 0. The sequence encoding
40 the VH-CH1 region (defined at the 5' and 3' edges by the oligonucleotides KSJ6 and 7 below) was PCR amplified from
Fab D1 .3 in pUC1 9 using oligonucleotides KSJ 6 and 7, which retain the Pst I site at the 5' end and introduce a Xho I
site at the 3' end, to facilitate cloning into fd CAT2. The sequences for the oligonucleotides KSJ6 and 7 are shown
below. The underlined region of KSJ7 shows the portion hybridising with the sequence for D1 .3.
45
KS J 6 : 5 1 AGG TGC AGC TGC AGG AGT CAG G 3 '
KS J7 : 5 * GGT GAC CTC GAG TGA AGA TTT GGG CTC AAC TTT C 3 '
PCR conditions were as described in example II, except that thirty cycles of PCR amplification were performed with
50 denaturation at 92°C for 45 seconds, annealing at 55°C for 1 minute and extension at 72°C for 1 minute. The template
used was DNA from TG1 cells containing Fab D1.3 in pUC19 resuspended in water and boiled. The template DNA
was prepared from the colonies by picking some colony material into 1 OOjlxI of distilled H 2 0 and boiling for 10 mins.
1uJ of this mixture was used in a 20uJ PCR. This regime resulted in amplification of the expected fragment of approx-
imately 600bp. This fragment was cut with Pst I and Xho I, purified from an agarose gel and ligated into Pst 1/Xho
55 1 -cut fdCAT2. The PCR mixture was extracted with phenol/chloroform and ethanol precipitated (Sambrook at al. supra.)
before digestion with Pst1 and Xhol (New England Biolabs according to manufacturers recommendations. The fragment
was resolved on 1%. Tris-Acetate EDTA agarose gel (Sambrook at al. supra) and purified using Geneclean (BIO 101,
Geneclean, La Jolla, San Diego, California, USA) according to manufacturers recommendations.
31
EP0 774 511 A1
fd-CAT2 vector DNA was digested with Pst 1 and Xho 1 (New England Biolabs ) according to manufacturers rec-
ommendations, extracted with phenol/chloroform and ethanol precipitated (Sambrookat al. supra.).
75ng of Pst 1/Xho 1 -digested vector DNA was ligated to 40ng of PCR-amplified Pst1 /Xho l-digested hEGF-R
fragment in 12uJ of ligation buffer (66mM TrisHCI (pH7.6), 5mM MgCI 2 , 5mM dithiothreitol, (100|ug/ml bovine serum
5 albumin, 0.5mM ATP, 0.5mM Spermidine) and 40C units T4 DNA ligase (New England Biolabs) for 16 hours at 16°C.
Two uJ of the ligation mixture was transformed into 200uJ of competent E.coli MC1061 cells, plated on 2TY agar
containing 15|ag/ml tetracycline and incubated at 30°C for 20 hours. A portion of the ligation reaction mixture was
transformed into E.coli MC1061 (Available from, for example Clontech Laboratories Inc, Palo Alto, California) and
colonies identified by hybridisation with the oligonucleotide D1 .3CDR3A as described in example 10. The presence of
10 the VHCH1 gene fragment was likewise confirmed by PCR, using oligonucleotides KSJ6 and 7. A representative clone
was called fd CAT2VHCH1 D1.3. The heavy-chain was deleted from Fab D1.3 in pUC19 by Sph I cleavage of Fab
D1 .3 plasmid DNA. The pUC 1 9 2.7Kb fragment containing the light chain gene was purified from a TAE agarose gel,
and 1 0ng of this DNA self-ligated and transformed into competent E.coli TG 1 . Cells were plated on 2TY agar containing
ampicillin (1 00u.g/ml) and incubated at 30°C overnight. The resulting, colonies were used to make miniprep DNA (Sam-
15 brook at al. supra), and the absence of the heavy chain gene confirmed by digestion with Sph I and Hind III. A repre-
sentative clone was called LCD1.3 DHC.
An overnight culture of fd CAT2VHCH1 D1.3 cells was microcentrifuged at 13,000Xg for 10 minutes and 50uJ of
the supernatant containing phage particles added to 50uJ of an overnight culture of LCD1 .3 DHC cells. The cells were
incubated at 37°C for 10 minutes and plated on 2TY agar containing ampicillin (100|ag/ml) and 15u.g/ml tetracycline.
20 Phage were prepared from some of the resulting colonies and assayed for their ability to bind lysozyme as described
in example 6.
The results (Figure 1 1 ) showed that when the heavy and light chain Fab derivatives from the original antibody D1 .3
were present, the pAb bound to lysozyme. pAb expressing the fd VHCH1 fragment did not bind to lysozyme unless
grown in cells also expressing the light chain. This shows that a functional Fab fragment was produced by an association
25 of the free light chain with VHCH1 fragment fused to gene III and expressed on the surface of the pAb.
Example 8
Isolation of Specific, Desired Phage from a Mixture of Vector Phage.
30
The applicant purified pAb (D1 .3) (originally called fdTscFcDI .3 in example 2) from mixtures using antigen affinity
columns. pAb (D1 .3) was mixed with vector fd phage (see table 1 ) and approximately 1 0 1 2 phage passed over a column
of lysozyme-Sepharose (prepared from cyanogen bromide activated sepharose 4B (Pharmacia, Milton Keynes, Bucks,
UK.) according to the manufacturers instructions. TG1 cells were infected with appropriate dilutions of the elutes and
35 the colonies derived, were analysed by probing with an oligonucleotide that detects only the pAb (D1.3) see Table 1
and Fig. 12. A thousand fold enrichment of pAb(D1 .3) was seen with a single column pass. By growing the enriched
phage and passing it down the column again, enrichments of up to a million fold were seen.
Enrichment was also demonstrated using purely immunological criteria. For example, 10 12 phage (at a ratio of 1
pAb (D1.3) to 4 x 10 6 fdTPs/Bs) was subjected to two rounds of affinity selection, and then 26 colonies picked and
40 grown overnight. The phage was then assayed for lysozyme binding by ELISA (as example 6). Five colonies yielded
phage with lysozyme binding activities, see table 1 , and these were shown to encode the scFv (D1 .3) by PCR screening
(example 13, using 30 cycles of 1 minute at 92°C, 1 minute at 60°C, 1 minute at 72°C using CDR3PCR1 and oligo 3
(fig. 4) as primers).
Thus very rare pAbs can be fished out of large populations, by using antigen to select and then screen the phage.
45 in this example, affinity chromatography of pAbs and oligonucleotide probing were carried out as described below.
Approximately 10 12 phage particles in 1ml MPBS were loaded onto a 1 ml lysozyme-Sepharose affinity column
which had been prewashed in MPBS. The column was washed in turn with 10 ml PBS; then 10 ml 50 mM Tris-HCI,
500 mM Nad pH 7.5; then 10ml 50 mM Tris-HCI 500 mM NaCI pH 8.5; then 5 mis 50 mM Tris-HCI, 500 mM NaCI pH
9.5 (adjusted with triethylamine) and then eluted with 5 ml 100 mM triethylamine. The eluate was neutralised with 0.5
50 m sodium phosphate buffer pH 6.8 and the phage plated for analysis. For a second round of affinity chromatography,
the first column eluate was plated to about 30,000 colonies per petri dish. After overnight growth, colonies were then
scraped into 5 ml 2 x TY medium, and a 20 jlxI aliquot diluted into 1 0 ml fresh medium and grown overnight. The phage
was PEG precipitated as d'escribed above, resuspended in 1 ml MPBS and loaded onto the column, washed and
eluted as above.
55 Oligonucleotides sythesised:
CDR3PCR1 5 1 TGA GGA C(A or T) C(A or T) GC CGT CTA CTA CTG
TGC 3 f
32
EP0 774 511 A1
40 pmole of oligonucleotide VH1 FOR (Ward, E. S., at al (1989) Nature 341 , 544-546), specific to pAb (D1.3) was
phosphorylated with 100 u.Ci a-32P ATP, hybridised (1pmole/ml) to nitrocellulose filters at 67°C in 6 x saline sodium
citrate (SSC) Sambrookat al., supra, buffer for 30 minutes and allowed to cool to room temperature for 30 mins, washed
3 x 1 min at 60°C in 0.1 x SSC.
5
Example 9
Construction of pAb Expressing Anti-hapten Activity
10 Oxazolone is a hapten that is commonly used for studying the details of the immune response. The anti-oxazalone
antibody, NQ11 has been described previously (E. Gherardi, R. Pannell, C. Milstein, J. Immunol. Method 126 61 -68).
A plasmid containing the VH and VL gene of NQ1 1 was converted to a scFv form by inserting the BstEl I/Sacl fragment
of scFvDI .3 myc (nucleotides 432-499 of Fig. 5) between the VH and VL genes to generate pscFvNQH , the sequence
of which is shown in fig. 1 3. This scFv was cloned into the Pst1/Xhol site of FdTPs/Xh (as described earlier) to generate
15 pAb NQ11 has an internal Pst1 site and so it was necessary to do a complete digest of pscFvNQH with Xhol followed
by a partial digest with Pst1 ).
The specific binding of pAb NQ11 was confirmed using ELISA. ELISA plates were coated at 37°C in 50 mM
NaHC03 at a protein concentration of 200 uxj/ml. Plates were coated with either hen egg lysozyme (HEL), bovine
serum albumin (BSA), or BSA conjugated to oxazolone (OX-BSA) (method of conjugation in Makela O.,, Kartinen M.,
20 Pelkonen J.L.T., Karjalainen K. (1978) J. Exp. Med. 148 1644). Preparation of phage, binding to ELISA plates, washing
and detection was as described in example 6. Samples were assayed in duplicate and the average absorbance after
10 minutes presented in figure 14.
This result demonstrates that the pAb NQ11 binds the correct antigen. Figure 14 also shows that pAb D1.3 and
pAb NQ11 bind only to the antigen against which the original antibodies were raised.
25
Example 10
Enrichment of pAb D1 .3 from Mixtures of Other pAb by Affinity Purification
30 3 x 10 10 phage in 10 mis of PBSM at the ratios of pAb D1 .3 to pAb NQ11 shown in table 2 were passed over a 1
ml lysozyme Sepharose column. Washing, elution and other methods were as described in example 8 unless otherwise
stated. Eluates from the columns were used to infect TG1 cells which were then plated out. Colonies were probed with
a probe which distinguishes pAb D1 .3 from pAb NQ11 . The sequence of this oligonucleotide (D1 .3CDR3A) is:-
35
5 ' GTA GTC AAG CCT ATA ATC TCT CTC 3 '
Table 2 presents the data from this experiment. An enrichment of almost 1000 fold was achieved in one round and an
enrichment of over a million fold in two rounds of purification. This parallels the result described in example 8.
40
Example 11
Insertion of a Gene Encoding an Enzyme (Alkaline phosphatase) into fd-CAT2
45 As an example of the expression of a functional enzyme on the bacteriophage surface, the applicants have chosen
bacterial alkaline phosphatase, an enzyme that normally functions as a dimer (McCracken, S. and Meighen, E. , J. Biol.
Chem. 255, p2396-2404, (1980)). The oligonucleotides were designed to generate a PCR product with an Apa L1 site
at the 5' end of phoA gene and a Not 1 site at its 3' end, thus facilitating cloning into fd-CAT 2 to create a gene III fusion
protein. The oligonucleotides synthesised were:
50
phoAl:5* TAT TCT CAC AGT GCA CAA ACT GTT GAA CGG ACA CCA
GAA ATG CCT GTT CTG 3' and,
phoA2 : 5 ' ACA TGT ACA TGC GGC CGC TTT CAG CCC CAG AGC GGC
55 TTT C3 1
The sequence of the phoA gene is presented in Chang C. N. at al., Gene 44, p121-125 (1986). The plasmid amplified
(pEK86) contains an alkaline phosphate gene which differs from the sequence of Chang at al, by a mutation which
33
EP0 774 511 A1
converts arginine to alamine at position 166.
The PCR reaction was carried out in 1 OOjllI of 10 mM Tris/HC1 pH 8.3, containing 50 mM KCI, 5mMdNTP 2.5 mM
MgCI 2 , 0.01 % gelatin, 0.25 units/uJ of Tag polymerase (Cetus/Perkin Elmer) and 0.5|Lig/ml template. The template was
the pEK86 plasmid (described by Chaidaroglou at al., Biochemistry 27 p8338-8343, 1988). The PCR was carried out
s in a Techne (Techne, Duxford, Cambridge, UK) PHC-2 dri-block using thirty cycles of 1 min at 92°C, 2 min at 50°C, 3
min at 72°C.
The resultant product was extracted with phenol:chloroform, precipitated with ethanol, and the pellet dissolved in
35uJ water. Digestion with 0.3 units/uJ of Apa L1 was carried out in 1 50uJ volume according to manufacturers instructions
for two hours at 37°C. After heat inactivation of the enzyme at 65°C , NaCI was added to a final concentration of 150mM
10 and 0.4 units/uJ Not1 enzyme added. After incubation for 2 hours at 37°C, the digest was extracted with phenol:chlo-
roform and precipitated as above, before being dissolved in 30uJ of water. The vector fd-CAT2 was sequentially digested
with Apa L1 and Not1 according to the manufacturers instructions and treated with calf intestinal alkaline phosphatase
as described in example 2. The sample was extracted three times with phenol:chloroform, precipitated with ethanol
and dissolved in water. The ligations were performed with a final DNA concentration of 1 -2ng/uJ of both the cut fd-CAT2
15 and the digested PCR product. The ligations were transformed into competent TG1 cells and plated on 2xTY tet plates.
Identification of clones containing the desired insert was by analytical PCR performed using the conditions and primers
above, on boiled samples of the resulting colonies. The correct clone containing the phoA gene fused in frame to gene
III was called fd-phoAla 166. The sequence at the junction of the cloning region is given in figure 15.
20 Example 12
Measuring Enzyme Activity of Phage-enzvme
Overnight cultures of TG1 or KS272 (E.coli cells lacking phoA. Strauch K. L, and Beckwith J. PN AS 85 1576-1580,
25 1 988) cells containing either fd-phoAla 166 or fd-CAT2 were grown at 37°C in 2xTY with 15jag/ml tetracycline. Con-
centrated, PEG precipitated phage were prepared as described earlier. Enzyme assays (Malamy, M.H. and Horecker
B.L., Biochemistry 3, p1893-1897, (1964)) were carried out at 24°C in a final concentration of 1M Tris/HC1 pH 8.0,
1mM 4-nitrophenyl phosphate (Sigma), 1mM MgC12. 1 OOjllI of a two times concentrate of this reaction mixture was
mixed with 1 OOjlxI of the test sample in a 96 well plate. Absorbance readings were taken every minute for 30 minutes
30 at a wavelength of 405nm in a Titretek Mk 2 plate reader. Initial reaction rates were calculated from the rate of change
of absorbance using a molar absorbance of 17000 l/mol/cm.
Standard curves (amount of enzyme vs. rate of change of absorbance) were prepared using dilutions of purified
bacterial alkaline phosphatase (Sigma type 1 1 1) in 1 0mM Tris/HCI pH 8.0, 1 mM EDTA. The number of enzyme molecules
in the phage samples were estimated from the actual rates of change of absorbance of the phage samples and com-
35 parison to this standard curve.
The results in Table 3 show that alkaline phosphatase activity was detected in PEG precipitated material in the
sample containing fd-phoAla166 but not fd-CAT2. Furthermore, the level of activity was consistent with the expected
number of 1 -2 dimer molecules of enzyme per phage. The level of enzyme activity detected was not dependent on the
host used for growth. In particular, fd-phoAla166 grown on phoA minus hosts showed alkaline phosphatase activity.
40 Therefore, the phage expressed active alkaline phosphatase enzyme, from the phoA-gene III fusion, on the phage
surface.
Example 1 3
45 Insertion of Binding Molecules into Alternative Sites in the Phage
The availability of an alternative site in the phage forthe insertion of binding molecules would open up the possibility
of more easily expressing more than one binding molecule e.g. an antibody fragment in a single pAb. This may be
used to generate single or multiple binding specificities. The presence of two distinct binding activities on a single
50 molecule will greatly increase the utility and specificity of this molecule. It may be useful in the binding of viruses with
a high mutational rate such as human immunodeficiency virus. In addition, it may be used to bring antigens into close
proximity (e.g. drug targetting or cell fusion) or it may act as a "molecular clamp" in chemical, immunological or enzy-
matic processes.
The vector fd-tet and the derivatives described here, have a single BamH1 site in gene 3. This has previously been
55 used for the expression of peptide fragments on the surface of filamentous bacteriophage (Smith GP. (1 985) Science
228 p1 31 5-1 31 7 and de la Cruz at al . (1988) J Biol. Chem. 263 p431 8-4322). This provides a potential alternative site
for the insertion of antibody fragments.
DNA fragments encoding scFv's from D1.3 or NQ11 were generated by PCR using the primers shown below.
34
EP0 774 511 A1
These primers were designed to generate a fragment with BamH1 sites near both the terminii, to enable cloning into
the BamHI site of gene3 (see figure 16(1)). The oligonucleotides used, also ensure that the resulting PCR product
lacks Pst1 and Xhol restriction sites normally used for manipulating the scFv's (see figure 16(1)). This will facilitate
subsequent manipulation of a second antibody fragment in the usual way at the N terminus of gene 3. The oligonucle-
5 otides used were:-
G3Baml 5 1 TTT AAT GAG GAT CCA CAG GTG CAG CTG CAA GAG 3 '
G3Bam2 5 ' AAC GAA TGG ATC CCG TTT GAT CTC AAG CTT 3 ' -
10 Preparation of vector and PCR insert
The PCR reaction was carried out in an 80 uJ reaction as described in example 11 using 1 ng/jixl of template and
0.25U/uJ of Taq polymerase and a cycle regime of 94°C for 1 minute, 60°C for 1 minute and 70°C for 2 minutes over
30 cycles. The template was either pscFvNQH (example 9) or scFvD1.3 myc (example 2). Reaction products were
15 extracted with phenol:chloroform, precipitated, dissolved in water and digested with BamHI according to manufacturers
instructions. The digest was re-extracted with phenol: chloroform, precipitated and dissolved in water.
The vector fdTPs/Xh was cleaved with BamHI and treated with calf intestinal phosphatase and purified as de-
scribed in example 2. Ligations were set up at a vector concentration of approximately 6ng/uJ and a PCR insert con-
centration of approximately 3ng/uJ. These were ligated for 2.5 hours at room temperature before transforming into
20 competent TG1 cells and plating on TYtet plates. The resultant colonies were probed as described in example 8. DNA
was prepared from a number of colonies and the correct orientation and insert size confirmed by restriction digestion
with Hind III in isolation or in combination with BamHI . (One Hind III site is contributed by one of the primers and the
other by the vector).
Two clones containing a D1.3 insert (fdTBaml) and fdTBam2) and one containing an NQ11 insert (NQUBaml)
25 were grown up and phage prepared as described earlier. ELISAs were carried out as described in example 6. No
specific signal was found for any of these clones suggesting that the natural BamHI site is not a suitable site for
insertion of a functional antibody (results not shown).
It may be possible to clone into alternative sites to retain binding activity. The peptide repeats present in gene III
may provide such a site (figure 16 blocks A and B). This can be done by inserting a BamHI site and using the PCR
30 product described above. To facilitate this, the natural BamHI site was removed by mutagenesis with the oligonucleotide
G3mut8Bam shown below (using an in vitro mutagenesis kit (Amersham International)):-
G3mut6Bam 5* CA AAC GAA TGG GTC CTC CTC ATT A 3 ?
35
The underlined residue replaces an A residue, thereby removing the BamHI site. DNA was prepared from a number
of clones and several mutants lacking BamHI sites identified by restriction digestion.
The oligonucleotide G3 Bamlink was designed to introduce a BamHI site at a number of possible sites within the
peptide linker sites A and B, see figure 16(2). The sequence of the linker is:
40
Bamlink 5 1 CC ( G or A ) CC ACC CTC GGA TCC ( G or A ) CC ACC
CTC 3 1
45 its relationship to the peptide repeats in gene III is shown in figure 16.
Example 14
PCR Assembly of Mouse VH and VL Kappa (VLK) Repertoires for Phage Display
50
The principle is illustrated in figure 17. Details are provided in sections A to F below but the broad outline is first
discussed.
1 . cDNA is prepared from spleen RNA from an appropriate mouse and the VH and VLK repertories individually
55 amplified. Separately, primers reverse and complementary to VH1 FOR-2 (domain 1 ) and VLK2BACK (domain 2)
are used to amplify an existing scFv-containing DNA by PCR. (The term FOR refers to e.g. a primer for amplification
of sequences on the sense strand resulting in antisense coding sequences. The term BACK refers to e.g. a primer
for amplification of sequences on the antisense strand resulting in sense coding sequences). This generates a
35
EP0 774 511 A1
'linker' molecule encoding the linker with the amino acid sequence (1 letter code) (GGGGS) 3 which overlaps the
two primary (VH and VLK) PCR products.
2. The separate amplified VH, VLK and linker sequences now have to be assembled into a continuous DNA mol-
ecule by use of an 'assembly' PCR. In the secondary 'assembly.' PCR, the VH, VLK and linker bands are combined
5 and assembled by virtue of the above referred to overlaps. This generates an assembled DNA fragment that will
direct the expression of VH and one VLK domain. The specific VH/VLK combination is derived randomly from the
separate VH and VLK repertoires referred to above.
The assembly PCR is carried out in two stages. Firstly, 7 rounds of cycling with just the three bands present in the
10 PCR, followed by a further 20 rounds in the presence of the flanking primers VH1 BACK (referring to domain 1 of VH)
and VLKFOR. The nucleotide sequences for these oligonucleotide primers are provided under the section entitled
'Primer Sequences' below. This two stage process, avoids the potential problem of preferential amplification of the first
combinations to be assembled.
For cloning into the phage system, the assembled repertoires must be 'tagged' with the appropriate restriction
15 sites. In the example provided below this is illustrated by providing an ApaL1 restriction site at the VH end of the
continuous DNA molecule and a Not 1 site at the VLK end of the molecule. This is carried out by a third stage PCR
using tagged primers. The nucleotide sequences for these oligonucleotide primers are also provided under the section
entitled 'Primer Sequences' below. There are however, 4 possible kappa light chain sequences (whereas a single
consensus heavy chain sequence can be used). Therefore 4 oligonucleotide primer sequences are provided for VLK.
20 For this third stage PCR, sets of primers which create the new restriction site and have a further 10 nucleotides
on the 5' side of the restriction site have been used. However, long tags may give better cutting, in which case 15-20
nucleotide overhangs could be used.
Scrupulously clean procedures must be used at all times to avoid contamination during PCR. Negative controls
containing no DNA must always be included to monitor for contamination. Gel boxes must be depurinated. A dedicated
25 Geneclean kit (B10 101, Geneclean, La Jolla, San Diego, California, USA) can be used according to manufacturers
instructions to extract DNA from an agarose gel. The beads, Nal and the NEW wash should be aliquoted.
All enzymes were obtained from CP Laboratories, P.O. Box 22, Bishop's Stortford, Herts CM20 3DH and the man-
ufacturers recommended and supplied buffers were used unless otherwise stated.
30 A. RNA Preparation
RNA can be prepared using may procedures well known to those skilled in the art. As an example, the following
protocol (Triton X-100 lysis, phenol/SDS RNase inactivation) gives excellent results with spleen and hybridoma cells
(the addition of VRC (veronal ribosyl complex) as an RNase inhibitor is necessary for spleen cells). Guanidinium iso-
35 thiocyanate/CsC1 procedures (yielding total cellular RNA) also give good results but are more time-consuming.
1 . Harvest 1 to 5 x 1 0 7 cells by centrif ugation in a bench tope centrifuge at 800xg for 1 0 minutes at 4°C. Resuspend
gently in 50ml of cold PBS buffer. Centrifuge the cells again at 800xg for 1 0 minutes at 4°C, and discard supernatant.
2. On ice, add 1 ml ice-cold lysis buffer to the pellet and resuspend it with a 1 ml Gilson pepette by gently pepetting
40 up and down. Leave on ice for 5 minutes.
3. After lysis, remove cell debris by centrif uging at 1 300 rpm for 5 minutes in a microfuge at 4°C, in precooled tubes.
4. Transfer 0.5 ml of the supernatant to each of two eppendorfs containing 60uJ 1 0% (w/v) SDS and 250 jlxI phenol
(previously equilibrated with 100 mM Tris-HCI pH 8.0). Vortex hard for 2 minutes, then microfuge (1 3000 rpm) for
five minutes at room temperature. Transfer the upper, aqueous, phase to a fresh tube.
45 5. Re-extract the aqueous upper phase five times with 0.5 ml of phenol.
6. Precipitate with 1/10 volume 3M sodium acetate and 2.5 volumes ethanol at 20°C overnight or dry ice-isopro-
panol for 30 minutes.
7. Wash the RNA pellet and resuspended in 50 jlxI to check concentration by OD260 and check 2 jLxg on a 1%
agarose gel. 40jag of RNA was obtained from spleen cells derived from mice.
50
Lysis buffer is [10mM Tris-HCI pH 7.4, 1mM MgC12, 150mM NaCI, 10mM VRC (New England Biolabs), 0.5% (w/v)
Triton X-100], prepared fresh.
Lysis buffer is [10mM Tris-HCI pH 7.4, 1mM MgCI 2 , 150mM NaCI, 10mM VRC (New England Biolabs), 0.5% (w/
v) Triton X-100], prepared fresh.
55
B. cDNA Preparation
cDNA can be prepared using many procedures well known to those skilled in the art. As an example, the following
36
EP0 774 511 A1
protocol can be used:
1 . Set up the following reverse transcription mix:
|llI
H 2 0 (DEPC-treated)
20
5mM dNTP
10
1 0 x first strand buffer
10
0.1M DTT
10
FOR primer(s) (10 pmol/uJ)
2
(each) (see below)
RNasin (Promega; 40 U/jllI)
4
NB
15
i) DEPC is diethylpyrocarbonate, the function of which is to inactivate any enzymes that could degrade DNA
or RNA
ii) dNTP is deoxynucleotide triphosphate
iii) DTT is dithiothreitol the function of which is as an antioxidant to create the reducing environment necessary
20 for enzyme function.
iv) RNasin is a ribonuclease inhibitor obtained from Promega Corporation, 2800 Woods Hollow Road, Madison,
Wisconsin, USA.
2. Dilute 1 0 jLxg RNA to 40 jlxI final volume with DEPC-treated water. Heat at 65°C for 3 minutes and hold on ice for
25 one minute ( to remove secondary structure).
3. Add to the RNA the reverse transcription mix (58 uJ) and 4 uJ of the cloned reverse transcriptase 'Super RT'
(Anglian Biotech Ltd. , Whitehall House, Whitehall Road, Colchester, Essex) and incubate at 42°C for one hour.
4. Boil the reaction mix for three minutes, cool on ice for one minute and then spin in a microfuge to pellet debris.
Transfer the supernatant to a new tube.
30
10 x first strand buffer is [1.4M KCI, 0.5M Tris-HCI pH 8.1 at 42°C 80mM MgCI 2 ].
The primers anneal to the 3' end. Examples of kappa light chain primers are MJK1 FONX, MJK2FONX, MJK4FONX
and MJK5FONX (provided under 'Primer Sequences' below) and examples of heavy chain primers are MIGG1 , 2 (CTG
GAC AGG GAT CCA GAG TTC CA) and MIGG3 (CTG GAC AGG GCT CCA TAG TTC CA) which anneal to CH1 .
35 Alternatively, any primer that binds to the 3' end of the variable regions VH, VLK, 'VL, or to the constant regions
CH1 , CK or CL can be used.
C. Primary PCRs
40 For each PCR and negative control, the following reactions are set up (e.g. one reaction for each of the four VLKs
and four VH PCRs). In the following, the Vent DNA polymerase sold by (CP Laboratories Ltd (New England Biolabs)
address given above) was used. The buffers are as provided by CP Laboratories.
JLXl
H 2 0
32.5
10 x Vent buffer
5
20 x Vent BSA
2.5
5mM dNTPs
1.5
FOR primer 10 pmol/uJ)
2.5
BACK primer 10pmol/uJ
2.5
The FOR and BACK primers are given in the section below entitled 'Primer Sequences'. For VH, the FOR primer is
VH1 FOR-2 and the BACK primer is VH1 BACK. For VLK the FOR primers are MJK1 FONX, MJK2FONX, MJK4FONX
55 and MJK5FONX (for the four respective kappa light chains) and the BACK primer is VK2BACK. Only one kappa light
chain BACK primer is necessary, because binding is to a nucleotide sequence common to the four kappa light chains.
UV this mix 5 minutes. Add 2.5 jllI cDNA preparation (from B above), 2 drops paraffin oil (Sigma Chemicals. Poole,
Dorset, UK). Place on a cycling heating block, e.g. PHC-2 manufactured by Techne Ltd. Duxford UK, preset at 94°C
37
EP0 774 511 A1
Add 1 jlxI Vent DNA polymerase under the paraffin. Amplify using 25 cycles of 94°C 1 min, 72°c 2 min. Post-treat at
60°C for 5 min.
Purify on a 2% Imp (low melting point agarose/TAE (tris-acetate EDTA)gel and extract the DNA to 20 uJ H 2 0 per
original PCR using a Geneclean kit (see earlier) in accordance with the manufacturers instructions.
D. Preparation of linker
Set up in bulk (e.g. 1 0 times)
H 2 0
34.3
10 x Vent buffer
5
20 x Vent BSA
2.5
5mM dNTPs
2
LINKFOR primer 10 pmol/uJ)
2.5
LINKBACK primer 10pmol/uJ
2.5
DNA from fcFv D1 .3 (example 2)
1
Vent enzyme
0.2
The FOR and BACK primers are given in the section below entitled 'Primer Sequences'. The FOR primer is LINKFOR
and the BACK primer is LINKBACK. Cover with paraffin and place on the cycling heating block (see above) at 94°C.
Amplify using 25 cycles of 94°C 1 min, 65°C 1 min, 72°C 2 min. Post-treat at 60°C for 5 min.
Purify on 2% 1mp/TAE gel (using a loading dye without bromophenol blue as a 93bp fragment is desired) and
elute with SPIN-X column (Costar Limited. 205 Broadway, Cambridge, Ma. USA.,) and precipitation. Take up in 5 jlxI
H 2 0 per PCR reaction.
E. Assembly PCRs
A quarter of each PCR reaction product (5uJ) is used for each assembly. The total volume is 25ul.
For each of the four VLK primers, the following are set up:
H 2 0
4.95
10 x Vent buffer
2.5
20 x Vent BSA
1.25
5mM dNTPs
0.8
UV irradiate this mix for 5 min. Add 5uJ each of Vh and VK band from the primary PCRs and 1 .5 uJ of linker as isolated
from the preparative gels and extracted using the Geneclean kit as described in C and D above. Cover with paraffin.
Place on the cycling heating block preset at 94°C. Add 1ul Vent under the paraffin. Amplify using 7 cycles of 94°C 2
min, 72°C 4 min. Then return the temperature to 94°C.
Add 1.5uJ each of VH1 BACK and the appropriate VKFOR primers MJK1FONX, MJK2FONX, MJK4FONX or
MJK5FONX (10 pmol/uJ) at 94°C. The primers should have been UV-treated as above. Amplify using 20 cycles of 94°C
1 .5 min, 72°C 2.5 min. Post-treat at 60°C for 5 min. Purify on 2% Imp/TAE gel and extract the DNA to 20uJ H 2 0 per
assembly PCR using a Geneclean kit (see earlier) in accordance with the manufacturers instructions.
F. Adding Restriction Sites
For each assembly and control set up:
H 2 0
36.5
10 x Taq buffer
5
5mM dNTPs
2
FOR primer (10 pmol/uJ)
2.5
BACK primer (10 pmol/uJ)
2.5
38
EP0 774 511 A1
(continued)
Assembly product
1
The FOR and BACK primers are given in the section below entitled 'Primer Sequences". The FOR primer is any of
JK1NOT10, JK2NOT10, JK4NOT10 or JK5NOT10 (for the four respective kappa light chains) for putting a Not1 re-
striction site at the VLK end. The BACK primer is HBKAPA10 for putting an Apal_1 restriction site at the VH end.
Cover with paraffin and place on the cycling heating block preset at 94°C. Add 0.5 jlxI Cetus Tag DNA polymerase
(Cetus/perkin-Elmer, Beaconsfield, Bucks, UK) under the paraffin. Amplification is carried out using 11 to 15 rounds
of cycling (depends on efficiency) at 94°C 1 min, 55°C 1 min, 72°C 2 min. Post-treat at 60°C for 5 min.
1 0 x Taq buffer is [0. 1 M Tris-HCI pH 8.3 at 25°C, 0.5M KCI, 1 5mM MgCI 2 , 1 mg/ml gelatin].
G. Work-up
Purify once with CHCI 3 /I AA (isoamylalcohol), once with phenol, once with CHCI 3 /IAA and back-extract everything
to ensure minimal losses. Precipitate and wash twice in 70% EtOH. Dissolve in 70uJ H 2 0.
Digest overnight at 37°C with Notl:
DNA (joined seq)
70
NEB Notl buffer x 10
10
NEB BSAx 10
10
Notl (10 U/jllI)
10
The DNA (joined sequence) above refers to the assembled DNA sequence comprising in the 5' to 3' direction
Apal_1 restriction site
VH sequence
Linker sequence
VLK sequence
Not 1 restriction site.
The VLK sequence may be any one of four possible kappa chain sequences.
The enzymes Not 1 above, ApaL1 below and the buffers NEB Not 1 , NEB BSA above and the NEB buffer 4 (below)
are obtainable from CP Laboratories, New England Biolabs mentioned above.
Re-precipitate, take up in 80uJ H 2 0. Add to this 10uJ NEB buffer 4 and 1 OjllI Apal 1.
Add the enzyme ApaL1 in aliquots throughout the day, as it has a short half-life at 37°C.
Purify on 2% 1mp/TAE gel and extract the DNA using a Geneclean kit, in accordance with the manufacturers
instructions. Redigest if desired.
H. Final DNA product
The final DNA product is an approximate 700 bp fragment with Apa L1 and Notl compatible ends consisting of
randomly associated heavy and light chain sequences linked by a linker. Atypical molecule of this type is the scFvDI .3
molecule incorporated into fdscFcD1.3 described in example 3. These molecules can then be ligated into suitable fd
derived vectors, e.g. fdCAT2 (example 5), using standard techniques.
Primer sequences
Primary PCR oligos (restrictions sites underlined):
39
EP0 774 511 A1
VH1FOR-2 TGA GGA GAC GGT GAC C GT GGT CCC TTG GCC CC
VH1BACK AGG TSM ARC TGC AGS AGT CWG G
MJK1FONX CCG TTT GAT TTC CAG CTT GGT GCC
MJK2FONX CCG TTT TAT TTC CAG CTT GGT CCC
MJK4FONX CCG TTT TAT TTC CAA CTT TGT CCC
MJK5FONX CCG TTT CAG CTC CAG CTT GGT CCC
VK2BACK GAC ATT GAG CTC ACC CAG TCT CCA
Ambiguity codes M - A or C, R = A or G, S = G or C, W = A or T
PCR oligos to make linker:
LINKFOR
LXNKBACK
TGG
GGG
AGA
ACC
CTC
ACG
GGT
GTC
GAG
ACC
CTC
GTC
AAT
TCC
GTC
TCA
For adding restriction sites:
HBKAPA10
CAT
GAC
CAC
AGT
GCA
CAG
GTS
MAR
CTG
CAG
SAG
TCW
JKINOT10
GG
GAG
TCA
TTC
TGC
CGC
i: i
GAT
TTC
CAG
CTT
JK2NOT10
GGT
GAG
GCC
TCA
TTC
TGC
GGC
CGC
CCG
- i A
TAT
Lao
JK4NOT10
GGT
CCC
TCA
rp «-n /->
TGC
/*"•
*n m /n
m m
•» <»
i-r-
JK5NOT10
GAG
CCC
TCA
rp rpr»
TGC
r~*
f-i r~+
CCG
" ^ ' fTl
-» a* ^
CAG
/•"» •* *-~*
i-fl «~r»
GGT
CCC
Example 15
Insertion of the Extracellular Domain of a Human Receptor for Platelet Derived Growth Factor (PDGF) soform BB into
fd CAT2
A gene fragment encoding the extracellular domain of the human receptor for platelet derived growth factor isoform
BB (h-PDGFB-R) was isolated by amplification, using the polymerase chain reaction, of plasmid RP41 , (from the Amer-
ican Type Culture collection, Cat. No. 50735), a cDNA clone encoding amino-acids 43 to 925 of the PDGF-B receptor
(Gronwald, R.G.K. etal PNAS 85 p3435-3439 (1 988)). Amino acids 1 to 32 of h-PDGFB-R constitute the signal peptide.
The oligonucleotide primers were designed to amplify the region of the h-PDGFB-R gene corresponding to amino acids
43 to 531 of the encoded protein. The primer RPDGF3 for the N-terminal region also included bases encoding amino
acids 33 to 42 of the h-PDGFB-R protein (corresponding to the first ten amino acids from the N-terminus of the mature
protein) to enable expression of the complete extracellular domain. The primers also incorporate a unique ApaL1 site
at the N-terminal end of the fragment and a unique Xhol site at the C terminal end to facilitate cloning into the vector
fdCAT2. The sequence of the primers is:
RPDGF3 5' CAC AGT GCA CTG GTC GTC ACA CCC CCG GGG CCA GAG
CTT GTC CTC AAT GTC TCC AGC ACC TTC GTT CTG 3 '
RPDGF2 5 ' GAT CTC GAG CTT AAA GGG CAA GGA GTG TGG CAC 3 '
PCR amplification was performed using high fidelity conditions (Eckert, K.A. and Kunkel, T.A. 1990 Nucl Acids
Research 18 3739-3744). The PCR mixture contained: 20mM TrisHCI (pH7.3 at 70°C, 50mM KC1, 4mM magnesium
chloride, 0.01% gelatin, 1mM each of dATP, dCTP, dGTP and dTTP, 500ng/ml RP41 DNA, 1|um each primer and 50
units/ml Tag polymerase (Cetus/Perkin Elmer, Beaconsfield, Bucks, U.K.). Thirty cycles of PCR were performed with
denaturation at 92°C for 1 min, annealing at 60°C for 1min and extension at 72°C for 1.5 min. This reaction resulted
in amplification of a fragment of ca. 1500bp as expected.
fdCAT2 vector DNA (see example 5) was digested with Apal_1 and Xhol (New England Biolabs) according to
40
EP0 774 511 A1
manufacturers recommendations, extracted with phenol/chloroform and ethanol precipitated (Sambrookat al, supra).
Cloning of amplified RP41 DNA into this vector and identification of the desired clones was performed essentially as
in example 7 except that digestion of the PCR product was with ApaL1 and Xho 1 . Colonies containing h-PDGFB-R
DNA were identified by probing with 32p labelled RPDGF2 and the presence of an insert in hybridising colonies was
s confirmed by analytical PCR using RPDGF3 and RPDGF2 using the conditions described in example 7.
Example 16
Binding of 1 25I-PDGF-BB to the Extracellular Domain of the Human Receptor for Platelet Derived Growth Factor
10 Isoform BB Displayed on the Surface of fd Phage. Measured using an Immunoprecipitation Assay.
Phage particles, expressing the extracellular domain of the human platelet derived growth factor isoform BB re-
ceptor (fd h-PDGFB-R), were prepared by growing E.coli MC1061 cells transformed with fd h-PDGFB-R in 50ml of
2xTY medium with 15ug/ml tetracycline for 16 to 20 hours. Phage particles were concentrated using polyethylene
15 glycol as described in example 6 and resuspended in PDGF binding buffer (25mM HEPES, pH7.4, o.15mM NaCI, 1 mM
magnesium chloride, 0.25% BSA) to 1/33rd of the original volume. Residual bacteria and undissolved material were
removed by spinning for 2 min in a mocrocentrifuge. Immunoblots using an antiserum raised against gene III protein
(Prof. I . Rashed, Konstanz, Germany) show the presence in such phage preparations of a genel I l-h-PDGFB-R protein
of molecular mass 125000 corresponding to a fusion between h-PDGFB-R external domain (55000 daltons) and genel II
20 (apparent molecular mass 70000 on SDS-polyacrylamide gel).
Duplicate samples of 35uJ concentrated phage were incubated with 125 I-PDGF-BB (78.7fmol, 70nCi, 882Ci/mmol;
Amersham International pic, Amersham, Bucks) for 1 hour at 37°C. Controls were included in which fdTPs/Bs vector
phage (figure 4) or no phage replaced fd h-BDGFB-R phage. After this incubation, 1 OjlxI of sheep anti-M13 polyclonal
antiserum (a gift from M. Hobart) was added and incubation continued for 30 min at 20°C. To each sample, 40ul (20ul
25 packed volume) of protein G Sepharose Fast Flow (Pharmacia, Milton Keynes) equilibrated in PDGF binding buffer
was added. Incubation was continued for 30 min at 20°C with mixing by end over end inversion on a rotating mixer.
The affinity matrix was spun down in a microcentrifuge for 2 min and the supernatant removed by aspiration. Non-
specifically bound 125 I-PDGF-BB was removed by resuspension of the pellet in 0.5ml PDGF binding buffer, mixing by
rotation for 5 min, centrifugation and aspiration of the supernatant, followed by two further washes with 0.5ml 0.1%
30 BSA, 0.2% Triton-X-100. The pellet finally obtained was resuspended in 1 0Oul PDGF binding buffer and counted in a
Packard gamma counter. For displacement studies, unlabelled PDGF-BB (Amersham International) was added to the
stated concentration for the incubation of 125 I-PDGF-BB with phage.
I 25 I-PDGF-BB bound to the fd h-PDGFB-R phage and was immunoprecipitated in this assay. Specific binding to
receptor phage was 3.5 to 4 times higher than the non-specific binding with vector phage fdTPs/Bs or no phage (fig.
35 19). This binding of 125 I-PDGF-BB could be displaced by the inclusion of unlabelled PDGF-BB in the incubation with
phage at 37°C (fig. 20). At 50nM, unlabelled PDGF-BB the binding of 125 I-PDGF-BB was reduced to the same level
as the fdTPs/Bs and no phage control. Figure 21 shows the same data, but with the non-specific binding to vector
deducted.
These results indicate that a specific saturable site for 125 I-PDGF-BB is expressed on fd phage containing cloned
40 h-PDGFB-R DNA. Thus, the phage can display the functional extracellular domain of a cell surface receptor.
Example 17, Construction of Phagemid Containing Genelll fused with the Coding Sequence for a Binding Molecule
It would be useful to improve the transfection efficiency of the phage-binding molecule system and also to have
45 the possibility of displaying different numbers and specificities of binding molecules on the surface of the same bacte-
riophage. The applicants have devised a method that achieves both aims.
The approach is derived from the phagemid system based on pUC119 [Vieira, J and Messing, J. (1987) Methods
Enzymol. 153:3]. In brief, gene III from fd-CAT2 (example 5) and gene III scFv fusion from fd-CAT2 scFv D1 .3 (example
2) were cloned downstream of the lac promoter in separate samples of pUC11 9, in order that the inserted gene III and
50 gene III fusion could be 'rescued' by M13M07 helper phage [Vieira, J and Messing, J. at supra.] prepared according
to Sambrootz et al. 1 989 supra. The majority of rescued phage would be expected to contain a genome derived from
the pUC119 plasmid that contains the binding molecule-gene III fusion and should express varying numbers of the
binding molecule on the surface up to the normal maximum of 3-5 molecules of gene III of the surface of wild type
phage. The system has been exemplified below using an antibody as the binding molecule.
55 An fdCAT2 containing the single chain Fv form of the D1.3 antilysozyme antibody was formed by digesting
fdTscFvDI .3 (example 2) with Pst1 and Xhol, purifying the fragment containing the scFv fragment and ligating this into
Pst1 and Xhol digested fdCAT2. The appropriate clone, called fdCAT2 scFvDI .3 was selected after plating onto 2xTY
tetracycline (15|Lig/ml) and confirmed by restriction enzyme and sequence analysis.
41
EP0 774 511 A1
Gene III from fd-CAT2 (example 5) and the gene III scFv fusion from fd-CAT2 scFcDI .3 was PCR-amplified using
the primers A and B shown below:
Primer A: TGC GAA GCT TTG GAG CCT TTT TTT TTG GAG ATT TTC
AAC G
Primer S: C AG TGA ATT CCT ATT AAG ACT CCT TAT TAC GCA GTA
w TGT TAG C
Primer A anneals to the 5' end of gene III including the ribosome binding site is located and incorporates a Hind
III site. Primer B anneals to the 3' end of gene III at the C-terminus and incorporates two UAA stop codons and an
EcoR1 site. 100 ng of fd-CAT2 and fd-CAT2 scFv D1 .3 DNA was used as templates for PCR-amplification in a total
15 reaction volume of SOjllI as described in example 7, except that 20 cycles of amplification were performed: 94°C 1
minute, 50°C 1 minute, 72°C 3 minutes. This resulted in amplification of the expected 1.2Kb fragment from fd-CAT2
and a 1 .8Kb fragment from fd-CAT2 scFv D1 .3.
The PCR fragments were digested with EcoR1 and Hind III, gel-purified and ligated into Eco-R1- and Hind Ill-cut
and dephosphorylated pUC119 DNA and transformed into E.coli TG1 using standard techniques (Sambrook et al., et
20 supra). Transformed cells were plated on SOB agar (Sambrook et al. 1989 supra) containing 100|ug/ml ampicillin and
2% glucose. The resulting clones were called pCAT-3 (derived from fd-CAT2) and pCAT-3 scFv D1 .3 (derived from fd-
CAT2 scFv D1.3).
Example 18. Rescue of Anti-Lysozyme Antibody Specificity from pCAT-3 scFv D1.3 by M13KQ7
25
Single pCAT-3 and pCAT-3 scFv D1 .3 colonies were picked into 1 .5ml 2TY containing 1 00|Lig/ml ampicillin and 2%
glucose, and grown 6 hrs at 30°C. 30uJ of these stationary cells were added to 6mls 2YT containing 1 00|ag/ml ampicillin
and 2% glucose in 50ml polypropylene tubes (Falcon, Becton Dickinson Labware, 1950 Williams Drive, Oxnard, CA.
USA) and grown for 1.5 hrs at 30°C at 380rpm in a New Brunswick Orbital Shaker (New Brunswick Scientific Ltd.,
30 Edison House 163 Dixons Hill road, North Mimms , Hatfield, UK). Cells were pelleted by centrifugation at 5,000g for
25 minutes and the tubes drained on tissue paper. The cell pellets were then suspended in 6mls 2TY containing
1.25x10 9 p.f.u. ml -1 M13K07 bacteriophage added. The mixture was left on ice for 5 minutes followed by growth at
35°C for 45 minutes at 450rpm. A cocktail was then added containing 4ul 100|ug/ml ampicillin, O.SjlxI 0.1M IPTG and
50uJ 10mg/ml kanamycin, and the cultures grown overnight an 35°C, 450rpm.
35 The following day the cultures were centrifuged and phage particles PEG precipitated as described in example 6.
Phage pellets were resuspended in 1 OOjllI TE (tris-EDTA see example 6) and phage titred on E.coli TG1. Aliquots of
infected cells were plated on 2TY containing either 1 00|ag/ml ampicillin to select for pUC1 1 9 phage particles, or SOjug/
ml kanamycin to select for the M1 3 K07 helper phage. Plates were incubated overnight at 37°C and antibiotic-resistant
colonies counted:
40
DNA
amp R
kan R
pCAT-3
1 .8x1 0 11 colonies
1 .2x1 0 9 colonies
pCAT-3scFv D1.3
2.4x1 0 11 colonies
2.0x1 0 9 colonies
This shows that the amp R phagemid particles are infective and present in the rescued phage population at a
100-fold excess over kan R M13K07 helper phage.
Phage were assayed for anti-lysozyme activity by ELISA as described in example 6, with the following modifica-
tions:
50
1 ) ELISA plates were blocked for 3 hrs with 2% Marvel/PBS.
2) SOjlxI phage, 400uJ 1xPBS and 50uJ 20% Marvel were mixed end over end for 20 minutes at room temperature
before adding 1 SOjlxI per well.
3) Phage were left to bind for 2 hours at room temperature.
4) All washes post phage binding were:
DO
2 quick rinses PBS/0.5% Tween 20
3x2 minute washes PBS/0.5% Tween 20
42
EP0 774 511 A1
2 quick rinses PBS no detergent
3x2 minute washes PBS no detergent
The result of this ELISA is shown in figure 22, which shows that the antibody specificity can indeed be rescued
5 efficiently.
It is considered a truism of bacterial genetics that when mutant and wild-type proteins are co-expressed in the
same cell, the wild-type proteins are co-expressed in same cell, the wild-type protein is used preferentially. This is
analogous to the above situation wherein mutant (i.e. antibody fusion) and wild-type gene III proteins (from M13K07)
are competing for assembly as part of the pUC11 9 phagemid particle. It is therefore envisaged that the majority of the
10 resulting pUC 119 phage particles will have fewer gene Ill-antibody fusion molecules on their surface than is the case
for purely phage system described for instance in example 2. Such phagemid antibodies are therefore likely to bind
antigen with a lower avidity than fd phage antibodies with three or more copies of the antibody fusion on their surfaces
(there is no wild-type gene III, in the system described, for instance, in example 2), and provide a route to production
of phage particles with different numbers of the same binding molecule (and hence different acidities for the ligand/
15 antigen) or multiple different binding specificities on their surface, by using helper phage such as M13K07 to rescue
cells expressing two or more gene Ill-antibody fusions.
It is also possible to derive helper phage that do not encode a functional gene III in their genomes (by for example
deleting the gene 1 1 1 sequence or a portion of it or by incorporating an amber mutation within the gene). These defective
phages will only grow on appropriate cells (for example that provide functional gene III in trans, or contain an amber
20 supressor gene), but when used to rescue phage antibodies, will only incorporate the gene III antibody fusion encoded
by the phagemid into the released phage particle.
Example 19. Transformation Efficiency of pCAT-3 and pCAT-3 scFv D1 .3 phagemids
25 pUC 19, pCAT-3 and pCAT-3 scFv D1.3 plasmid DNAs, and fdCAT-2 phage DNA was prepared, and used to
transform E.coli TG1 , pCAT-3 and pCAT-3 scFv D1 .3 transformations were plated on SOB agar containing 100uxj/ml
ampicillin and 2% glucose, and incubated overnight at 30°C. fdCAT-2 transformations were plated on TY agar con-
taining 15|ag/ml tetracycline and incubated overnight at 37°C. Transformation efficiencies are expressed as colonies
per |ug of input DNA.
30
DNA
Transformation efficiency
pUC 19
1.10 9
pCAT-3
1.10 8
pCAT-3scFv D1.3
1.10 8
fd CAT-2
8.10 5
As expected, transformation of the phagemid vector is approximately 100-fold more efficient that the parental
fdCAT-2 vector. Furthermore, the presence of a scFv antibody fragment does not compromise efficiency. This improve-
ment in transformation efficiency is practically useful in the generation of phage antibodies libraries that have large
repertoires of different binding specificities.
Example 20
PCR Assembly of a Single Chain Fv Library from an Immunised Mouse
To demonstrate the utility of phage for the selection of antibodies from repertoires, the first requirement is to be
able to prepare a diverse, representative library of the antibody repertoire of an animal and display this repertoire on
the surface of bacteriophage fd.
Cytoplasmic RNA was isolated according to example 1 4 from the pooled spleens of five male Balb/c mice boosted
8 weeks after primary immunisation with 2-phenyl-5-oxazolone (ph OX) coupled to chicken serum albumin. cDNA
preparation and PCR assembly of the mouse VH and VL kappa repertoires for phage display was as described in
example 14. The molecules thus obtained were ligated into fdCAT2.
Vector fdCAT2 was extensively digested with Not1 and Apal_1. ; purified by electroelution (Sambrook at al.a989
supra) and 1 uxj ligated to 0.5 jLxg (5 uxj for the hierarchial libraries: see example 22) of the assembled scFv genes in
1 ml with 8000 units T4 DNA ligase (New England Biolabs). The ligation was carried out overnight at 16°C. Purified
ligation mix was electroporated in six aliquots into MC1061 cells (W. J. Dower, J. F. Miller & C. W. Ragsdale Nucleic
Acids Res. 16 6127-6145 1988) and plated on NZY medium (Sambrook at al. 1989 supra) with 15u.g/ml tetracycline,
43
EP0 774 511 A1
in 243x243 mm dishes (Nunc): 90-95% of clones contained scFv genes by PCR screening.
Recombinant colonies were screened by PCR (conditions as in example 7 using primers VH1 BACK and MJK1 FONX,
MJK2FONX, MJK4FONX and MJK5FONX (see example 14) followed by digestion with the frequent cutting enzyme
BstNl (New England Biolabs, used according to the manufacturers instructions). The library of 2x1 0 5 clones appeared
5 diverse as judged by the variety of digestion patterns seen in Figure 23, and sequencing revealed the presence of
most VH groups (R. Dildrop, Immunol. Today 5 85-86. 1984) and VK subgroups (Kabat. E.A. at al. 1987 supra) (data
not shown). None of the 568 clones tested bound to phOx as detected by ELISA as in example 9.
Thus the ability to select antibody provided by the use of phage antibodies (as in example 21 ) is essential to readily
isolate antibodies with antigen binding activity from randomly combined VH and VL domains. Very extensive screening
10 would be required to isolate antigen-binding fragments if the random combinatorial approach of Huse at al. 1 989 (supra)
were used.
Example 21
15 Selection of Antibodies Specific for 2-phenyl-5-oxazolone from a Repertoire Derived from an Immunised Mouse
The library prepared in example 20 was used to demonstrate that ability of the phage system to select antibodies
on the basis of their antibody specificity.
None of the 568 clones tested from the unselected library bound to phOx as detected by ELISA.
20 Screening for binding of the phage to hapten was carried out by ELISA: 96-well plates were coated with 10 jag/ml
phOx-BSA or 10 |ag/ml BSA in phosphate-buffered saline (PBS) overnight at room temperature. Colonies of phage-
transduced bacteria were inoculated into 200 uJ 2 x TY with 1 2.5 uxj/ml tetracycline in 96-well plates ('cell wells', Nuclon)
and grown with shaking (300 rpm) for 24 hours at 37°C. At this stage cultures were saturated and phage titres were
reproducible (10 10 TU/ml). 50 uJ phage supernatant, mixed with 50 jlxI PBS containing 4% skimmed milk powder, was
25 then added to the coated plates. Further details as in example 9.
The library of phages was passed down a phOx affinity column (Table 4A), and eluted with hapten. Colonies from
the library prepared in example 22 were scraped into 50ml 2 x TY medium 37 and shaken at 37°C for 30 min. Liberated
phage were precipitated twice with polyethylene glycol and resuspended to 10 12 TU (transducing units)/ml in water
(titred as in example 8). For affinity selection, a 1 ml column of phOx-BSA-Sepharose (O. Makela, M. Kaartinen, J.L.
30 t. Pelonen and K. Karjalainen J. Exp. Med. 148 1644-1660, 1978) was washed with 300 ml phosphate-buffered saline
(PBS), and 20 ml PBS containing 2% skimmed milk powder (MPBS). 10 12 TU phage were loaded in 10 ml MPBS,
washed with 10 ml MPBS and finally 200 ml PBS. The bound phage were eluted with 5 ml 1 mM 4-e-amino-caproic
acid methylene 2-phenyloxazol-5-one (phOx-CAP; O. Makela at al. 1978, supra). About 10 6 TU eluted phage were
amplified by infecting 1 ml log phase E.coli TG1 and plating as above. For a further round of selection, colonies were
35 scraped into 10 ml 2 x TY medium and then processed as above. Of the eluted clones, 13% were found to bind to
phOx after the first round selection, and ranged from poor to strong binding in ELISA.
To sequence clones, template DNA was prepared from the supernatants of 1 0 ml cultures grown for 24 hours, and
sequenced using the dideoxy method and a Sequenase kit (USB), with primer LINKFOR (see example 14) for the VH
genes and primer fdSEQI (5'-GAA TTT TCT GTA TGA GG) for the Vk genes. Twenty-three of these hapten-binding
40 clones were sequenced and eight different VH genes (A to H) were found in a variety of pairings with seven different
Vk genes (a to g) (Fig. 24). Most of the domains, such as VH-B and Vk-d were 'promiscuous 1 , able to bind hapten with
any of several partners.
The sequences of the V-genes were related to those seen in the secondary response to phOx, but with differences
(Fig. 24). Thus phOx hybridomas from the secondary response employ somatically mutated derivatives of three types
45 of Vk genes - Vkoxl. 'Vkox-like' and Vk45.1 genes (C. Berek, G. M. Griffiths & C. Milstein Nature 316 412-418 (1985).
These can pair with VH genes from several groups, from Vkoxl more commonly pairs with the VHoxl gene (VH group
2. R. Dildrop uupra). Vkoxl genes are always, and Vkox-like genes often, found in association with heavy chains (in-
cluding VHoxl) and contain a short five residue CDR3, with the sequence motif Asp-X-Gly-X-X in which the central
glycine is needed to create a cavity for phOx. In the random combinatorial library however, nearly all of the VH genes
50 belonged to group 1 , and most of the Vk genes were ox-like and associated with VH domains with a five residue CDR3,
motif Asp/Asn-X-Gly-X-X (Fig. 24). Vkoxl and VHoxl were found only once (Vk-f and VH-E), and not in combination
with each other. Indeed Vk-f lacks the Trp91 involved in phOx binding and was paired with a VH (VH-C) with a six
residue CDR3.
A matrix combination of VH and VK genes was identified in phOx-binding clones selected from this random com-
55 binational library. The number of clones found with each combination are shown in Fig. 25. The binding to phOx-BSA,
as judged by the ELISA signal, appeared to vary (marked by shading in Fig. 25). No binding was seen to BSA alone.
A second round of selection of the original, random combinational library from immune mice resulted in 93% of
eluted clones binding phOx (Table 4). Most of these clones were Vk-d combinations, and bound strongly to phOx in
44
EP0 774 511 A1
ELISA (data not shown). Few weak binders were seen. This suggested that affinity chromatography had not only
enriched for binders, but also for the best.
Florescence quench titrations determined the Kd of VH-B/Vk-d for phOx-GABA as 1 0" 8 M (example 23), indicating
that antibodies with affinities representative of the secondary response can be selected from secondary response, only
5 two (out of eleven characterised) secrete antibodies of a higher affinity than VH-B/Vk-d (C. Berek at al. 1985 supra).
The- Kd of VH-B/Vk-b for phOx-GABA was determined as 10 -5 M (example 23). Thus phage bearing scFv fragments
with weak affinities can be selected with antigen, probably due to the avidity of the nultiple antibody heads on the phage.
This example shows that antigen specificities can be isolated from libraries derived from immunised mice. It will
often be desired to express these antibodies in a soluble form for further study and for use in therapeutic and diagnostic
10 applications. Example 23 demonstrates determination of the affinity of soluble scFv fragments selected using phage
antibodies. Example 27 demonstrates that soluble fragments have similar properties to those displayed on phage. For
many purposes it will be desired to construct and express an antibody molecule which contains the Fc portions of the
heavy chain, and perhaps vary the immunoglobulin isotype. To accomplish this, it is necessary to subclone the antigen
binding sites identified using the phage selection system into a vector for expression in mammalian cells, using meth-
15 odology similar to that described by Orlandi, R. at al. (1989, supra). For instance, the VH and VL genes could be
amplified separately by PCR with primers containing appropriate restriction sites and inserted into vectors such as
pSV-gpt HulgGI (L. Riechmann at al Nature 332 323-327), 1988) which allows expression of the VH domain as part
of a heavy chain IgGI isotype and pSV-hyg HuCK which allows expression of the VL domain attached to the K light
chain constant region. Furthermore, fusions of VH and VL domains can be made with genes encoding non-immu-
20 noglobulin proteins, for example, enzymes.
Example 22
Generation of Further Antibody Specificities by the Assembly of Hierarchical Libraries
25
Further antibody specificities were derived from the library prepared and screened in examples 20 and 21 using
a hierarchical approach.
The promiscuity of the VH-B and Vk-d domains prompted the applicants to force further pairings, by assembling
these genes with the entire repertoires if either Vk or VH genes from the same immunised mice. The resulting 'hierar-
30 chical 1 libraries, (VH-B x Vk-rep and VH-rep x Vk-d), each with 4x1 0 7 members, were subjected to a round of selection
and hapten-binding clones isolated (Table 4). As shown by ELISA, most were strong binders. By sequencing twenty-
four clones from each library, the applicants identified fourteen new partners for VH-B and thirteen for Vk-d (Fig. 24).
Apart from VH-B and Vk-c, none of the previous partners (or indeed other clones) from the random combinatorial library
was isolated again. Again the Vk genes were mainly ox-like and the VH genes mainly group 1 (as defined in Dildrop,
35 R. 1 984 supra), but the only examples of Vkoxl (Vk-h, -p, -q and -r) have Trp91 , and the VH-CDR3 motif Asp-X-Gly-
X-X now predominates. Thus some features of the phOx hybridomas seemed to emerge more strongly in the hierarchial
library. The new partners differed from each other mainly by small alterations in the CDRs, indicating that much of the
subtle diversity had remained untapped by the random combinatorial approach. More generally it has been shown that
a spectrum of related antibodies can be made by keeping one of the partners fixed and varying the other, and this
40 could prove invaluable for fine tuning of antibody affinity and specificity.
Therefore, again, phage antibodies allow a greater range of antibody molecules to be analysed for desired prop-
erties.
This example, and example 21, demonstrate the isolation of individual antibody specificities through display on
the surface of phage. However, for some purposes it may be more desirable to have a mixture of antibodies, equivalent
45 to a polyclonal antiserum (for instance, for immunoprecipitation). To prepare a mixture of antibodies, one could mix
clones and express soluble antibodies or antibody fragments or alternatively select clones from a library to give a highly
enriched pool of genes encoding antibodies or antibody fragments directed against a ligand of interest and express
antibodies from these clones.
so Example 23
Selection of Antibodies Displayed on Bacteriophage with Different Affinities for 2-phenyl-5-oxazolone using Affinity
Chromatography
55 The ELISA data shown in example 21 suggested that affinity chromatography had not only enriched for binders,
but also for the best. To confirm this, the binding affinities of a strong binding and a weak binding phage were determined
and then demonatrated that they could be separated from each other using affinity chromatography.
Clones VH-B/Vk-b and VH-B/Vk-d were reamplified with MJK1FONX, MJK2FONX, MJK4FONX and MJK5FONX
45
EP0 774 511 A1
(see example 14) and VH1 BACK-Sfil (5'-TCG C GG CCC AGC CGG CC A TGG CC(G/C) AGG T(C/G)(A/C) A(A/G)C
TGC AG(C/G) AGT C(A/T)G G), a primer that introduces an Sfil site (underlined) at the 5' end of the VH gene. VH-B/
Vk-d was cloned into a phagemid e.g. pJM1 (a gift from A. Griffiths and J. Marks) as an Sfil-Notl cassette, downstream
of the pelB leader for periplasmic secretion (M. Better at al. supra), with a C-terminal peptide tag for detection (see
5 example 24 and figure), and under th control of a P L promoter (H. Shimatake & M. Rosenberg Nature 292 128-132
1981). The phagemid should have the following features: a) unique Sfil and Not1 restriction sites downstream of a
pelB leader; b) a sequence encoding a C-terminal peptide tag for detection; and c) a X P L promoter controlling expres-
sion. 10 litre cultures of E.coli N4830-1 (M. E. Gottesman, S. Adhya & A. Das J.Mol.Biol 140 57-75 1980) harbouring
each phagemid were induced as in K. Nagai & H. C. Thogerson (Methods Enzymol 1 53 461 -481 1 987) and supernatants
10 precipitated with 50% ammonium sulphate. The resuspended precipitate was dialysed into PBS + 0.2 mM EDTA (PB-
SE), loaded onto a 1.5ml column of phOx:Sepharose and the column washed sequentially with 100 ml PBS: 100 ml
0. 1 M Tris-HCI, 0.5 M NaCI, pH 8.0: 1 0ml 50 mM citrate, pH 5.0: 1 0 ml 50 mM citrate, pH4.0, and 20 ml 50 mM glycine,
pH 3.0. scFv fragments were eluted with 50 mM glycine, pH 2.0, neutralised with Tris base and dialysed against PBSE.
VH-B/Vk-b was cloned into a phagemid vector based on pUC119 encoding identical signal and tag sequences to pJMI,
15 and expression induced at 30°C in a 10 litre culture of E.coli TG1 harbouring the phagemid as in D. de Bellis & I.
Schwartz (1 980 Nucleic Acids Res 1 8 1 31 1 ). The low affinity of clone VH-B/Vk-b made its purification on phOx-Sepha-
rose impossible. Therefore after concentration by ultrafiltration (Filtron, Flowgen), the supernatant (100 ml of 600 ml)
was loaded onto a 1 ml column of protein A-Sepharose cpoupled (E. Harlow & D. Lane 1 988 supra) to the monoclonal
antibody 9E10 (Evan, G. I. at al. Mol.Cell Biol.5 3610-3616 1985) that recognises the peptide tag. The column was
20 washed with 200 ml PBS and 50 ml PBS made 0.5 M in NaCI. scFv fragments were eluted with 100 ml 0.2M glycine,
pH 3.0, with neutralisation and dialysis as before.
The Kd (1 .0 ± 0.2 x 1 0 -8 M) for clone VH-B/Vk-d was determined by fluorescence quench titration with 4-E-amino-
butyric acid methylene 2-phenyl-oxazol-5-one (phOx-GABA Co. Makela et al, 1978 supra). Excitation was at 280 nm,
emission was monitored at 340 nm and the K d calculated. The K d of the low affinity clone VH-B/Vk-b was determined
25 as 1 .8± 0.3 x 10- 5 M (not shown). To minimise light adsorption by the higher concentrations of phOx-GABA required,
excitation was at 260 nm and emission was monitored at 304 nm. In addition the fluorescence values were divided by
those from a parallel titration of the lysozyme binding Fv fragment D1.3. The value was calculated as in H. N. Eisen
Meth.Med.Res. 10 115-121 1964. A mixture of clones VH-B/Vk-b and VH-B/Vk-d, 7x10 10 TU phage in the ratio 20 VH-
B/Vk-b : 1 VH-B/Vk-d were loaded onto a phOx-BSA-Sepharose column in 10 ml MPBS and eluted as above. Eluted
30 phage were used to reinfect E.coli TG1 , and phage produced and harvested as before. Approximately 10 11 TU phage
were loaded onto a second affinity column and the process repeated to give a total of three column passes. Dilutions
of eluted phage at each stage were plated in duplicate and probed separately with oligonucleotides specific for Vk-b
(5'G AG CGG GTA ACC ACT GTA CT) or Vk-d (5'-GAA TGG TAT AGT ACT ACC CT). After these two rounds, essentially
all the eluted phage were VH-B/Vk-d (table 4). Therefore phage antibodies can be selected on the basis of the antigen
35 affinity of the antibody displayed.
Example 24
Construction of Phagemid pHEN1 for the Expression of Antibody Fragments Expressed on the Surface of
40 Bacteriophage following Superinfection
The phagemid pHEN1 (figure 26) is a derivative of pUC119 (Vieira, J. & Messing, J. Methods Enzymol 153 pp
3-11, 1987). The coding region of g3p from fdCAT2, including signal peptide and cloning sites, was amplified by PCR,
using primers G3FUFO and G3FUBA (given below) (which contain EcoRI and Hindi 1 1 sites respectively), and cloned
45 as a Hindlll-EcoRI fragment into pUC119. The Hindll l-Notl fragment encoding the g3p signal sequence was the re-
placed by a pelB signal peptide (Better, M. at al. Science 240 1041-1043, 1988) with an internal Sfil site, allowing
antibody genes to be cloned as fil-Notl fragments. A peptide tag, c-myc, (Munro, S. & Pelham, H. Cell 46 291-300,
1986) was introduced directly after the Notl site by cloning an oligonucleotide cassette, and followed by an amber
codon introduced by site-directed mutagenesis using an in vitro mutagenesis kit (Amersham International) (figure 26b).
G3FUFO , 5 1 -CAG T GA ATT C TT ATT AAG ACT CCT TAT TAC GCA GTA
TGT TAG C;
G3FUBA, 5 1 -TGC G AA GCT T TG GAG CCT TTT TTT TTG GAG ATT TTC
AAC G;
46
EP0 774 511 A1
Example 25
Display of Single Chain Fv and Fab Fragments Derived from the Anti-Oxazolone Antibody NQ1 0. 1 2.5 on Bacteriophage
fd using pHEN1 and fdCAT2
5
A range of constructs (see figure 27) were made from a clone (essentially construct II in pUC19) designed for
expression in bacteria of a soluble Fab fragment (Better at al. 1988 see above) from the mouse anti-phOx (2-phenyl-
5-oxazolone) antibody NQ10.12.5 (Griffiths, G. M. at al. Nature 312, 271-275, 1984). In construct II, the V-regions are
derived from NQ10.12.5 and attached to human Ck and CH1 (y1 isotype) constant domains. The C-terminal cysteine
10 residues, which normally form a covalent link between light and heavy antibody chains, have been deleted from both
the constant domains. To clone heavy and light chain genes together as Fab fragments (construct II) or as separate
chains (constructs III and IV) for phage display, DNA was amplified from construct II by PCR to introduce a Notl re-
striction site at the 3' end, and at the 5' end either an ApaLI site (for cloning into fd-CAT2) or Sfil sie (for cloning into
pHEN1 ). The primers FABNOTFOK with VH1 BACKAPA (or VH 1 BACKSF1 1 5) were used for PCR amplification of genes
15 encoding Fab fragments (construct II), the primers FABNOTFOH with VH1 BACKAPA (or VH1 BACKSFI15) for heavy
chains (construct III), and the primers FABNOTFOK and MVKBAAPA (or MVKBASFI) for light chains (construct IV).
The single-chain Fv version of NQ10.12.5 (construct I) has the heavy (VH) and light chain (Vk) variable domains
joined by a flexible linker (Gly 4 Ser) 3 (Huston, J. S. at al. Proc. Natl. Acad. Sci. USA 85 5879-5883, 1988) and was
constructed from construct II by 'splicing by overlap extension' as in example 14. The assembled genes were reamplified
20 with primers VK3F2NOT and VH1 BACKAPA (or VH1 BACKSFI15) to append restriction sites for cloning into fd-CAT2
(ApaLI-Notl) or pHEN1 (Sfil-Notl).
VH1 BACKAPA, 5 1 -CAT GAC CAC A GT GCA CA G GT(C/G) (A/C)A(A/G)
CTG CAG (C/G)AG TC( A/T ) GG;
VH1BACKSFI15 , 5 r -CAT GCC ATG ACT CGC GGC CCA GCC GGC C AT
GGC C(C/G)A GGT (C/G)(A/C)A (A/G)CT GCA G(C/G)A GTC
( A/T )GG;
FABNOTFOH, 5 ! -CCA CGA TTC T GC GGC CGC TGA AGA TTT GGG CTC
AAC TTT CTT GTC GAC ;
FABNOTFOK, 5 ' -CCA CGA TTC T GC GGC CGC TGA CTC TCC GCG GTT
GAA GCT CTT TGT GAC;
MVKBAAPA, 5 1 -CAC A GT GCA C TC GAC ATT GAG CTC ACC CAG TCT
CCA ;
MVKBASFI , 5 1 -CAT GAC CAC GC G GCC CAG CCG GCC ATG GCC GAC
ATT GAG CTC ACC CAG TCT CCA;
VK3F2NOT, 5 * -TTC T GC GGC CGC CCG TTT CAG CTC GAG CTT GGT
ccc.
Restriction sites are underlined.
45 Rescue of Phage and Phagemid particles
Constructs l-IV (figure 27) were introduced into both fd-CAT2 and pHEN1. Phage fd-CAT2 (and fd-CAT2-1 , II, III or
IV) was taken from the supernatant of infected E.coli TG1 after shaking at 37°C overnight in 2xTY medium with 1 2.5u.g/
ml tetracycline, and used directly in ELISA. Phagemid pHEN1 (and pHEN1-l and II) in E.coli TG1 (supE) were grown
50 overnight in 2 ml 2xTY medium, 100 |ug/ml ampicillin, and 1% glucose (without glucose, expression of g3p prevents
later superinfection by helper phage). 1 OjlxI of the overnight culture was used to innoculate 2 ml of 2xTY medium, 1 0Ojug/
ml ampicillin, 1% glucose, and shaken at 37°C for 1 hour. The cells were washed and resuspended in 2xTY, 100 u.g/
ml ampicillin, and aphagemid particles rescued by adding 2 uJ (10 8 pfu) VCSM13 helper phage (Stratagene). After
growth for one hour, 4uJ kanamycin (25 mg/ml) was added, and the culture grown overnight. The phagemid particles
55 were concentrated 10-fold for ELISA by precipitation with polyethylene glycol.
47
EP0 774 511 A1
ELISA
Detection of phage binding to 2-phenyl-5-oxazolone (phOx) was performed as in example 9. 96-well plates were
coated with 10 jag/ml phOx-BSA or 10 jug/ml BSA in PBS overnight at room temperature, and blocked with PBSS
5 containing 2% skimmed milk powder. Phage (mid) supernatant (50 uJ) mixed with 50 uJ PBS containing 4% skimmed
milk powder was added to the wells and assayed. To detect binding of soluble scFv or Fab fragments secreted from
pHEN1, the c-myc peptide tag described by Munro and Pelham 1986 supra, was detected using the anti-myc mono-
clonal 9E 10 (Evan, G. I. etal. Mol Cell Biol 5 3610-3616, 1 985) followed by detection with peroxidase-conjugated goat
anti-mouse immonoglobulin. Other details are as in example 9.
10 The constructs in fdCAT2 and pHEN1 display antibody fragments of the surface of filamentous phage. The phage
vector, fd-CAT2 (figure 8) is based on the vector fd-tet (Zacher, A. N. at al. Gene 9 127-140, 1980) and has restriction
sites (ApaLI and Notl) for cloning antibody genes (or other protein) genes for expression as fusions to the N-terminus
of the phage coat protein g3p. Transcription of the antibody-g3p fusions in fd-CAT2 is driven from the gene III promoter
and the fusion protein targetted to the periplasm by means of the g3p leader. Fab abd scFv fragments of NQ10.12.5
15 cloned into fd-CAT2 for display were shown to bind to phOx-BSA (but not BSA) by ELISA (table 5). Phage were con-
sidered to be binding if A 405 of the sample was at least 10-fold greater that the background in ELISA.
The phagemid vector, pHEN1 (fig. 26), is based upon pUC119 and contains restriction sites (Sfil and Notl) for
cloning the fusion proteins. Here the transcription of antibody-g3p fusions is driven from the inducible lacZ promoter
and the fusion protein targetted to the periplasm by means of the pelB leader. Phagemid was rescued with VCSM13
20 helper phage in 2xTY medium containing no glucose or IPTG: under these conditions there is sufficient expression of
antibody-g3p. Fab and scFv fragments of NQ10.12.5 cloned into pHEN1 for display were shown to bind to phOx-BSA
(but not BSA) by ELISA (Table 5) using the same criterion as above.
An alternative methodology for preparing libraries of Fab fragments expressed on the surface of phage would be to:
25 1 . Prepare a library of phage expressing heavy chain (VHCH) genes from inserts in the phage genome.
2. Prepare a library of light chain genes in a plamid expression vector in E.coli, preferably a phagemid, and isolate
the soluble protein light chins expresed from this library.
3. Bind the soluble protein light chains fromt he library to the heavy chain library displayed on phage.
4. Select phage with the desired properties of affinity and specificity.
30 These will encode the heavy chain (VHCH) genes.
5. Isolate the light chain genes encoding ight chains which form suitable antigen binding sites in combination with
the selected heavy chains, preferably by using superinfectin of bacteria, containing phagemid expressing the light
chain, with phage expressing the selected heavy chain (as described in example 20) and then assaying for antigen
binding.
35
Example 26
Rescue of Phagemid Encoding a Gene III Protein Fusion with Antibody Heavy or Light Chains by Phage Encoding the
Complementary Antibody Chain Displayed on Phage and the Use of this Technique to Make Dual Combinatorial
40 Libraries
With random combinatorial libraries there is a limitation on the potential diversity of displayed Fab fragments due
to the transformation efficiency of bacterial cells. Described here is a strategy (dual combinatorial libraries) to overcome
this problem, potentially increasing the number of phage surveyed by a factor of 10 7 .
45 For assembly of heavy and light chains expresses from different vectors, phagemid (pHE N 1 -1 1 1 or IV) was grown
in E.coli HB2151 (a non-supressor strain) to allow production of soluble chains, and rescued as above (example 27)
except that helper phage were used expressing partner chains as fusions to g3p (10 9 TU fd-CAT2-IV or III respectively)
and 2 uJ tetracycline (12.5 mg/ml) in place of kanamycin.
50 Separate Vectors to Encode Fab Heavy and Light Chains
The heavy and light chains of Fab fragments can be encoded together in the same vector (example 25) or in
different vectors. To demonstrate this the heavy chain (construct III) was cloned into pHEN1 (to provide soluble frag-
ments) and the light chain (construct IV) intofd-CAT2 (to make the fusion with g3p). The phagemid pHEN 1 -III, grown
55 in E.coli HB2151 (non-supressor) was rescued with fd-CAT2-IV phage, and phage(mid) shown to bind to phOx:BSA,
but not to BSA (Table 5). This demonstrates that soluble light chain is correctly associating with the heavy chain an-
chored to the g3p, since neither heavy chain nor light chain alone bind antigen (Table 5).
Similar results were obtained in the reverse experiment (with phagemid pHEN-1 -IVandfd-CAT2-lll phage) in which
48
EP0 774 511 A1
the heavy chain was produced as a soluble molecule and the light chain anchored to g3p (Table 5). Hence a Fab
fragment is assembled on the surface of phage by fusion of either heavy or light chain to g3p, provided the other chain
is secreted using the same or another vector (figure 28).
The resulting phage population is a mixture of phage abd rescued phagemid. The ratio of the two types of particle
5 was assessed by infecting log phase E.coli TG1 and plating on TYE plates with either 15 jug/ml tetracycline (to select
forfd-CAT2) or 100 u.g/ml ampicillin (to select for pHEN1). The titre of fd-CAT2 phage was 5 x 10 11 TU/ml and the titre
of pHEN1 2 x 10 10 TU/ml, indicating a packaging ratio of 25 phage per phagemid.
Demonstrated here is an alternative strategy involving display of the heterodimeric antibody Fab fragments on the
surface of phage. One of the chains is fused to g3p and the other is secreted in soluble form into the periplasmic space
10 of the E.coli where it associates non-covalently with the g3p fusion, and binds specifically to antigen. Either the light
or heavy chain can be fused to the g3p: they are displayed on the phage as Fab fragments and bind antigen (Figure
28). Described are both phage and phagemid vectors for surface display. Phagemids are probably superior to phage
vectors for creation of large phage display libraries. Particularly in view of their higher transfection efficiencies (Two to
three orders of magnitude higher), allowing larger libraries to be constructed. The phagemid vector, pHEN1 also allows
15 the expression of soluble Fab fragments in non-suppressor E.coli.
Also demonstrated here is that heavy and light chains encoded on the same vector (construct II), or on different
vectors (constructs III and IV) can be displayed as Fab fragments. This offers two distinct ways of making random
combinatorial libraries for display. Libraries of heavy and light chain genes, amplified by PCR, could be randomly linked
by a 'PCR assembly' process (example 14) based on 'splicing by overlap extension', cloned into phage(mid) display
20 vectors and expressed from the same promoter as part of the same transcript (construct II) as above, or indeed from
different promoters as separate transcripts. Here the phage(mid) vector encodes and displays both chains. For a com-
binatorial library of 10 7 heavy chains and 10 7 light chains, the potential diversity of displayed Fab fragments (10 14 ) is
limited by the transfection efficiency of bacterial cells by the vector (about 10 9 clones per uxj cut and ligated plasmid
at best) (W.J. Dower at al Nucl. Acids. Res. 1 6 61 27-61 45, 1 988). Libraries thus prepared are analogous to the random
25 combinatorial library method described by Huse, W.D. et al Science 246 1275-1281 (1989), but have the important
additional feature that display on the surface of phage gives a powerful method of selecting antibody specificities from
the large number of clones generated.
Alternatively, libraries of heavy and light chains could be cloned into different vectors for expression in the same
cell, with a phage vector encoding the g3p fusion and a phagemid encoding the soluble chain. The phage acts as a
30 helper, and the infected bacteria produced both packaged phage and phagemid. Each phage or phagemid displays
both chains but encodes only one chain and thus only the genetic information for half of the antigen-binding site.
However, the genes for both antibody chains can be recovered separately by plating on the selective medium, sug-
gesting a means by which mutually complementary pairs of antigen binding heavy and light chain combinations could
be selected from random combinatorial libraries. For example, a light chain repertoire on fd phage could be used to
35 infect cells harbouring a library of soluble heavy chains on the phagemid. The affinity purified phagemid library could
then be used to infect E.coli, rescued with the affinity purified phage library, and the new combinatorial library subjected
to a further round of selection. Thus, antibody heavy and light chain genes are reshuffled after each round of purification.
Finally, after several rounds, infected bacteria could be plated and screened individually for antigen-binding phage.
Such 'dual' combinatorial libraries are potentially more diverse than those encoded on a single vector. By combining
40 separate libraries of 10 7 light chain phage(mid)s, the diversity of displayed Fab fragments (potentially 10 14 ) is limited
only by the number of bacteria (1 0 12 per litre). More simply, the use of two vectors should also facilitate the construction
of 'hierarchical' libraries, in which a fixed heavy or light chain is paired with a library or partners (example 22), offering
a means of 'fine-tuning' antibody affinity and specificity.
45 Example 27
Induction of Soluble scFv and Fab Fragments using Phagemid pHEN1
Further study of antibodies which have been expressed on the surface of phage would be greatly facilitated if it is
50 simple to switch to expression in solution.
E.coli HB2151 was infected with pHEN phagemid (pHEN1-l or II), and plated on YTE, 100|ag/ml ampicillin plates.
Colonies were shaken at 37°C in 2xTY medium, 100 |ag/ml ampicillin, 1% glucose to OD 55O =0.5 to 1.0. Cells were
pelleted, washed once in 2xTY medium, resuspended in medium with 100 uxj/ml ampicillin, 1 mM isopropyl (3-D-thi-
ogalactoside (IPTG), and grown for afurther 1 6 hours. Cells were pelleted and the supernatant, containing the secreted
55 chains, used directly in ELISA.
The phagemid pHEN1 has the advantage over phage fd-CAT2, in that antibody can be produced either for phage
display (by growth in supE strains of E.coli) or as a tagged soluble fragment (by growth in non-suppressor strains), as
a peptide tag (example 24) and amber codon were introduced between the antibody and g3p. Secretion of soluble Fab
49
EP0 774 511 A1
fragments from pHENI -II or scFv fragments from pHEN1-l was demonstrated after growth in E.coli HB2151 and in-
duction with IPTG using Western blots (Figure 29). For detection of secreted proteins, 1 OjllI supernatant of induced
cultures were subjected to SDS-PAGE and proteins transferred by electroblotting to Immobilon-P (Millipore). Soluble
heavy and light chain were detected with goat polyclonal anti-human Fab antiserum (Sigma) and peroxidase conjugated
5 rabbit anti-goat immunoglobulin (Sigma), each at- a dilution of 1 :1000. The tagged VK domain was detected with 9E10
antibody (1:1000) and peroxidase conjugated goat anti-mouse immunoglobulin (Fc specific) (1:1000) (Sigma) or with
a peroxidase labelled anti-human CK antiserum (Dako). 3,3'-diaminobenzidine (DAB;Sigma) was used as peroxidase
substrate (Harlow E., at al. 1988 Supr). With the scFv, the fragments were detected using the 9E10 anti-myc tag
antibody (data not shown). With the Fab, only the light chain was detected by 9E10 (or anti-human CK) antibody, as
10 expected, while the anti-human Fab antiserum detected both heavy and light chains. Binding of the soluble scFv and
Fab fragments to phOx-BSA (but not to BSA) was also demonstrated by ELISA (Table 5B). Thus scFv and Fab frag-
ments can be displayed on phage or secreted as soluble fragments from the same phagemid vector.
Example 28
15
Increased Sensitivity in ELISA assay of Lysozyme using FDTscFvDI .3 as Primary Antibody Compared to Soluble
scFvD1.3
In principle the use of phage antibodies should allow more sensitive immunoassays to be performed than with
20 soluble antibodies. Phage antibodies combine the ability to bind a specific antigen with the potential for amplification
through the presence of multiple (ca.2800) copies of the major coat protein (g8p) on each virion. This would allow the
attachment of several antibody molecules directed against M13 to each virion followed by the attachment of several
molecules of peroxidase-conjugated anti-species antibody (anti-sheep) IgG in the case below). Thus for every phage
antibody bound to antigen there is the potential for attaching several peroxidase molecules whereas when a soluble
25 antibody is used as the primary antibody this amplification will not occur.
ELISA plates were coated overnight at room temperature using 200jllI of 10 fold dilutions of hen egg lysozyme
(1000, 100, 10, 1, 0.1 and 0.01 |ug/ml) in 50mM NaHC0 3 , pH9.6. ELISA was performed as described in example 4
except that (i) incubation with anti-lysozyme antibody was with either FDTscFvDI .3 (pAb; 1 0 11 phage per well; 1 .6mol)
or soluble affinity purified scFvDI .3 (18jag per well; 0.7nmol) (ii) incubation with second antibody was with 1/100 dilution
30 of sheep anti-M13 serum for FDTscFvDI. 3 samples or with or 1/100 dilution of rabbit anti-scFvD1 .3 serum (from S.
Ward) for soluble scFvDI .3 samples (iii) peroxidase-conjugated rabbit anti-goat immunoglobulin (Sigma; 1/5000) was
used for FDTscFvDI .3 samples and peroxidase-conjugated goat anti-rabbit immunoglobulin (Sigma; 1/5000) was used
for soluble scFvD1.3 samples. Absorbance at 405nm was measured after 15h. The results are shown in Figures 30
and 31 . In these figures lysozyme concentrations for coating are shown on a log scale of dilutions relative to 1u.g/ml.
35 (i.e. log = -3 =1 mg/ml ; log = 2 = 0.01 |ag/ml)
Higher signals were obtained with FDTscFvDI .3 at all concentrations of lysozyme (Fig. 31 ) but the difference was
very marked at the greatest dilutions, where antigen quantities are most limiting (Figs. 30 and 31). This suggests that
phage antibodies may be particularly valuable for sandwich type assays where the capture of small amounts of antigen
by the primary antibody will generate an amplified signal when phage antibodies directed against a different epitope
40 are used as the second antigen binding antibody.
Example 29
Direct Rescue and Expression of Mouse Monoclonal Antibodies as Single Chain Fv Fragments on the Surface of
45 Bacteriophage fd.
The principle is very similar to that described in example 14. It consists of the PCR assembly of single chain
antibodies from cDNA prepared from mouse monoclonals. As an example, the rescue and expression of two such
antibodies from monoclonals expressing antibodies against the steroid hormone oestriol is described.
50
A. RNA Preparation
RNA can be prepared using many procedures well known to those skilled in the art. In this example, the use of
Triton X-100 lysis, phenol/SDS RNase inactivation gave excellent results.
55
1 . The mouse monoclonal cells that were used here had been harvested by centrifugation and resuspended in
serum free medium. They were then centrifuged and resuspended in saline and after a final centrifugation step,
resuspended in sterile water at 1 x 10 7 cells per ml. (Normally cells would be washed in PBS buffer and finally
50
EP0 774 511 A1
resuspended in PBS buffer, but these particular cells were supplied to us as described frozen in water.).
2. To 750jllI of cells was added 250ul of ice cold 4X lysis buffer (40mM Tris HCI pH 7.4/4mM MgCI 2 /600mM NaCI/
40mM VRC (Veronyl ribosyl complex)/2% Triton X-100). The suspension was mixed well and left on ice for 5
minutes.
5 3. Centrifugation was carried out at 4°C in a microfuge at 1 3000 rpm for 5 min.
The supernatant is then phenol extracted three times, phenol chloroform extracted three times and finally, ethanol
precipitated as described in the materials and methods. The precipitate was resuspended in 50ul water.
4. The optical density of the RNA at 260nm with a 2.5ul sample in 1 ml water was measured. The RNA was checked
by electrophoresis of a 2ug sample on a 1 % agarose gel. RNA in the range of 32ug to 42ug was obtained by this
10 method.
B. cDNA Preparation
The method used is the same as that described in example 14. Two cDNA preparations were made. These were
15 from RNA extracted from the monoclonals known as cell lines 013 and 014 which both express antibodies against eh
steroid hormone, oestriol.
C. Primary PCRs
20 The method used is essentially the same as that described in example 14. The VH region was amplified with the
primers VH1 BACK and VH1 FOR-2. For the Vkappa region, four separate reactions were carried out using the primer
VK2BACK and wither MJK1 FONX, MJK2FONX, MJK4FONX or MJK5FONX. Samples (5ul) were checked on a 1 .5%
agarose gel. From this it was observed that for cDNA prepared from the two oestriol monoclonals the primers VK2BACK
and MJK1FONX gave the best amplification of the Vkappa region. The VH bands and the Vkappa bands amplified
25 with VK2BACK/MJK1 FONX were purified on 2% low melting point agarose gels for each monoclonals. The DNA bands
were excised from the gel and purified using a dedicated Geneclean kit as described in example 14.
D. Preparation of linker
30 The method used is essentially the same as that described in example 14. In this case, the amplified linker DNA
was purified on a 2% agarose gel and recovered from the gel with a dedicated "Mermaid" kit (BIO 101 , Geneclean, La
Jolla, San Diego, California, USA) using the manufacturers instructions.
E. Assembly PCRs
35
The method used is essentially the same as that described in example 14. In this case, the assembled PCR product
was purified on a 2% agarose gel and recovered from the gel with a dedicated "Mermaid" kit.
F. Adding restriction sites and work-up
40
The assembled product was "tagged" with Apa LI and Not I restriction sites. The DNA was then digested with Apa
LI and Not I to give the appropriate sticky ends for cloning and then purified on a 2% low melting point agarose gel
and extracted using a Geneclean kit. The method used is the same as that described in example 14.
45 G. Cloning into Vector fd-CAT2
A total of 15ug of CsCI purified fd-CAT2 DNA was digested with 100 units of the restriction enzyme Not I (New
England Biolabs) in a total volume of 200ul 1X NEB Not I buffer with 1X NEB acetylated BSA for a total of 3 hours at
37°C. The vector DNA was the treated twice with 15ul Strataclean (a commercially available resin for the removal of
so protein), following the manufacturers instructions (Stratagene, 11099 North Torrey Pines Road, La Jolla, California,
USA). The DNA was then ethanol precipitated and redissolved in TE buffer (Sambrook at al., 1 989 supra). The DNA
was then digested with 1 00 units of the restriction enzyme Apa LI (New England Biolabs) in a total volume of 200ul 1 X
NEB Buffer 4 overnight at 37°C. The vector was then purified with a Chroma Spin 1000 column following the manu-
facturers instructions (Clontech Laboratories Inc, 4030 Fabian way, Palo Alto, California, USA). This step removes the
55 Apa Ll/Not I fragment to give cut vector DNA for maximum ligation efficiency.
Ligation reactions were carried out with 2.5-1 Ong of the DNA insert and 1 0ng of vector in a total volume of 1 0ul of
1X NEB ligase buffer with 1ul of NEB ligase (New England Biolabs) at 16°C overnight (approx 16 hours).
51
EP0 774 511 A1
H. Transformation and growth
E.coli strain TG1 was made competent and transformed with the fdCAT2 recombinant DNA as described by Sam-
brook at al, 1 989 Supra. The cells were plated out on LBtet plates (1 Og tryptone, 5g yeast extract, 1 0g NaCI, 1 5g bacto-
5 agar per litre with 15ug/ul of tetracycline added just before pouring the plates) and grown overnight.
Single well isolated colonies were then inoculated into 1 0 ml of LBtet broth (LB medium with 1 5ug/ul of tetracycline)
in 50 ml tubes. After overnight growth at 35°C/350rpm in a bench top centrifuge. The supernatants were transferred
to 15 ml centrifuge tubes and 2ml 20% PEG 8000/2. 5M NaCI added to each. After incubating at room temperature for
20-30 minutes, the recombinant phage was pelleted by centrifugation at 9000rpm in a Sorval SM24 rotor for 30 minutes.
10 The PEG supernatant was discarded. Any remaining PEG was removed with a pasteur pepette after a brief (2 minutes)
centrifugation step. This last step was repeated to make sure that no PEG remained. The phage pellet was then
resuspended in 500ul PBS buffer. This was transferred to a microcentrifuge tube and spun at 13000 rpm to remove
any remaining cells. The phage supernatant was transferred to a fresh tube.
15 I. Assay for antibody expression
Bacteriophage fd recombinants were screened for the expression of antibody against oestriol by ELISA. This
method is described in example 6. In this case the following alterations are relevant.
20 1 . Microtitre plates were coated overnight with 40ug/ml oestriol-6 carboxymethyloxime-BSA (Steraloids, 31 Rad-
cliffe Road, Croydon, CRO 5QJ, England).
2. 1st antibody was the putative phage anti oestriol antibody. 50ul of phage in a final volume of 200ul of sterile
PBS combining 0.25% gelatin was added to each well.
3. 2nd antibody was sheep anti M1 3 at 1 :1000 dilution.
25 4. 3rd antibody was peroxidase conjugated rabbit anti goat immunoglobulin.
Recombinants expressing functional antibody were detected by incubation with the chromogenic substrate 2'2'
axinobis (3-ethyl benzthiazoline sulphonic acid). The results are shown in figures 32 and 33.
30 Example 30
Kinetic Properties of Alkaline Phosphatase Displayed on the Surface of Bacteriophage fd
This example demonstrates that kinetic properties of an enzyme expressed on phage are qualitatively similar to
35 those in solution. Bacteriophage fd displaying alkaline phosphatase fusions of gene 3 with either the native arginine
(see example 31) or the mutant residue alanine at position 166 (see example 11) were prepared by PEG precipitation
as described in the materials and methods.
The kinetic parameters of alkaline phosphatase expressed on the surface of fd phage were investigated in 1M
Tris/HCI, pH8.0 at 20°C with 1ml 4-nitrophenyl phosphate as substrate. The reactions were initiated by the addition of
40 1 OOjlxI of a phage-alkaline phosphatase fusion preparation, 50 fold concentrated with respect to the original culture
supernatant. The rate of change of absorbance was monitored at 410nm using a Philips 8730 spectrophotometer and
the initial reaction rate calculated using a molar absorbance of 16200 1/mol/cm. For the fdphoAla 166 enzyme but not
fdphoArg166 a lag phage was seen following this addition, the reaction rate accelerating until a steady state was
obtained after approximately 60 to 90 sacs. This steady state rate was used for determination of kinetic parameters.
45 No deviation form Michaelis Menten kinetics was apparent for either phage enzyme, \folues of K m and k cat were derived
from plots of s/v against s and are shown in Table 6.
Because of the difficulty in establishing the relationship between the number of phage particles an the number of
active enzyme dimers formed on the phage k cat values are expressed not as absolute values, but as relative values
between the two enzyme forms. Western blots (carried out as in example 31 using antig3p antiserum) of the phage
so enzyme preparations used in this experiment showed approximately equal intensities for the full length fusion band
with the Arg1 66 and Ala1 66 enzymes when detected using antibody directed against gene3. In these preparations the
intact fusion represents approximately 30% of the detected material. The two preparations were therefore assumed
to be expressing approximately the same concentrations of intact fusions.
Table 6 summarises the kinetic data from this experiment and compares it with data from Chaidaroglou, A. at al
55 (Biochemistry 27, 8338-8343 (1988)) obtained with soluble preparations of the wild type and mutant enzyme forms.
The same substrate and assay conditions were used in both experiments. Soluble alkaline phosphatase was also
tested in parallel in our experiments (K m =8.5|ixM; kcat=3480 mol substrate converted mol enzyme -1 min -1 ).
The effect of mutating arginine at position 166 to alanine is qualitatively similar for the phage enzyme as for the
52
EP0 774 511 A1
soluble enzyme. K m is increased about 15 fold and the relative k cat is decreased to 36% of that for wild type. This
increased K m would reflect a reduction in substrate affinity in the phage enzyme on mutation of Arg1 66, as was proposed
for the soluble enzyme (Chaidaroglou at al, 1988 supra), assuming the same kinetic mechanism applies. There are,
however, some quantitative differences in the behaviour of K m of the phage enzyme. The K m of 73jaM observed for
5 fdphoArgl 66 compares with a K m of 1 2.7|llM for the free enzyme; the K m for fdphoAlal 66 is l070uJvl whereas the free
mutant enzyme has a K m of 1620|lxM. One can speculate that the higher K m for fdphoArg 166 and the lower K m for
fdphoAlal 66, compared to the soluble enzymes result from the 'anchored' alkaline phosphatase fusion molecules
interacting to form dimers in a different manner to the enzyme in free solution.
The relative values of k cat for the Arg1 66 and Ala1 66 forms are however very similar for both the phage enzymes
10 and the soluble enzymes, a reduction occurring on mutation to 35 to 40% of the value for the native enzyme. The rate
limiting step, determining k cat , for soluble phoArg166 is thought to be dissociation of non-covalently bound phosphate
from the enzyme (Hull W.E. at al. Biochemistry J_5, 1547-1561 1976). Chaidaroglou at al (1988) supra suggest that,
for the soluble enzyme, mutation of Arg166 to alanine alters additional steps, one of which may be hydrolysis of the
phosphoenzyme intermediate. The similarity in the reduction in on mutation of Arg166 to alanine for the phage
15 enzymes suggests that the same steps may be altered in a quantitatively similar manner in the mutant phage enzyme
as in the mutant soluble enzyme.
Thus, enzymes displayed on phage show qualitatively similar characteristics to soluble enzymes.
Example 31
20
Demonstration using Ultrafiltration that Cloned Alkaline Phosphatase Behaves as Part of the Virus Particle
The construct fdphoAlal 66 (derived in example 11) was converted back to the wild type residue (arginine) at
position 166 by in vitro mutagenesis (Amersham International) using the printer
25
AFARG16 6 : 5 ' TAGCATTTGCGCGAGGTCACA 3 ' .
This construct with the wild type insert was called fdphoArgl 66.
E.coli TG1 or KS272 cells (cells with a deletion in the endogenous phoA gene, Strauch and Beckwith, 1988 Supra)
containing either fd-phoAla166, fdphoArgl 66 or fd-CAT2 were grown for 16 hours at 37°C in 2xTY with 15u.g/ml tet-
racycline. Concentrated phage were prepared as follows. Phage-enzyme cultures are clarified by centrifugation (15
min at 10,000 rpm, 8 x 50 ml rotor, sorval RC-5B centrifuge). Phage are precipitated by adding 1/5 volume 20% poly-
ethylene glycol, 2.5 M Nacl, leaving for 1 hr at 4°C, and centrifuging (as above). Phage pellets are resuspended in 10
mM Tris-HCI, pH 8.0 to 1/1 00th of the original volume, and residual bacteria and aggregated phage removed by cen-
trifugation for 10 to 15 minutes in a bench microcentrifuge at 13000 rpm at 4°C.
SDS/Polyacrylamide gel electrophoresis and western blotting were basically as described previously (example 2).
Denatured samples consisting of 1 6jlxI of a 50 fold concentrate of phage were separated using a 10% SDS/polyacry-
lamide gel and detected with polyclonal antiserum raised against either E.coli alkaline phosphatase (Northumbria Bi-
ologicals, South Nelson Industrial Estate, Cramlington, Northumberland, NE23 9HL) or against the minor coat protein
encoded by gene 3 (from Prof. I. Rasched, Universitat Konstanz, see Stengele at al, 1990) at 1 in 1000 dilution. This
was followed by incubation with peroxidase-conjugated goat-anti-rabbit immunoglobulin (Sigma 1 in 5000) and detec-
tion with the ECL Western blotting system (Amersham International).
The presence of fusion proteins was confirmed by western blotting of proteins from phage particles derived from
fd-phoAla166 (phage-enzyme) or fd-CAT2 (vector phage). Detection with antiserum raised against the gene 3 protein
reveals a product of apparent relative molecular mass (Mr) of 63,000 in vector phage (figure 34e). Although this is
different from the predicted molecular weight based on the amino acid sequence (42,000), the natural product of gene
3 has previously been reported to exhibit reduced mobility during electrophoresis (Stengele at al, 1990).
In the fd-phoAla166 sample the largest band has an apparent Mr of 115,000, (fig. 34). Taking into account the
aberrant mobility of the gene 3 portion of the fusion, this is approximately the size expected from fusing with an alkaline
phosphatase domain of 47 kD. This analysis also reveals that a proportion of the Gene3 reactive material in this phage-
enzyme preparation is present at the size of the native gene3 product, suggesting that degradation is occurring. In the
preparation shown in figure 34, approximately 5-10% of the gene 3 fusions are intact. In more recent preparations and
in all the preparations used in this example and example 32, approximately 30-60% of fusions are full length.
The protein of Mr 1 1 5,000 is the major protein observed in Western blots of phage-enzyme derived from TG 1 cells
when probed with antiserum raised against E.coli alkaline phosphatase (anti-BAP), confirming the assignment of this
band to intact fusion. Further, when phage enzyme is prepared using KS272 cells, which have a deletion in the en-
dogenous p_hoA gene (Strauch & Beckwith, 1988, supra.) it is also the major band. There are additional bands at Mr
53
EP0 774 511 A1
95000 and 60000 reactive with anti-BAP antiserum which may indicate degradation of the fusion product.
The anti-BAP antiserum also reacts wit material running with the dye front and with a molecule of Mr 45,000 but
evidence suggests that this material is not alkaline phosphatase. This pattern is detected in PEG precipitated vector
phage samples (figure 34c) and is not therefore contributed by protein expressed frcm the cloned phoA gene. These
5 bands are detected in culture supernatants of cells carrying fd-CAT2 but is not detected in the supernatant of uninfected
cells (not shown) and so either represents cross-reactivity with phage encoded material or with a PEG precipitable
cellular component leaked from infected cells (Boeke atal, Mol. Gen. Genet. 186 , 185-192 1982). Although the fragment
of Mr, 45,000 is close to the size of free alkaline phosphatase (47,000), it is present in phage preparations from KS272
cells which have a deletion in the phoA locus. Furthermore its mobility is different from purified alkaline phosphatase
10 and they can be distinguished by electrophoresis (figure 34d).
Ultrafiltration was used to confirm that the fusion protein behaved as though it were part of a larger structure, as
would be expected for an enzyme bound to a phage particle. Phage samples (1 OOjlxI of a 50 fold concentrate) were
passed through ultrafiltration filters with a nominal molecular weight limit of 300000 daltons (Ultrafree-MC filters, Mill-
ipore) by centrif ugation for 5 to 1 5 minutes at 1 3,000 r.p.m. in an MSE microcentaur microf uge. Retained material was
15 recovered by resuspending in 1 OOjllI of 10mM Tris, pH 8.0.
Phage-enzyme or free alkaline phosphatase (83ng) mixed with vector phage were passed through filters with a
nominal molecular weight limit of 300,000 daltons (Ultrafree-MC filters, Millipore). Figure 35 A again shows that the
band of Mr, 115,000 is the major product reactive with anti-BAP antiserum. This and the other minor products reactive
with anti-BAP are present in material retained by the ultrafiltration membrane. Analysis of retained and flow through
20 fractions of phage preparations derived from KS272 demonstrates that different molecular species are being separated
by the ultrafiltration membranes. Figure 35b shows the protein of Mr 115,000 is retained by the filter whereas the
putative degradation products of Mr 95,000 and 60,000 found in phage preparations derived from KS272 cells, are not
retained.
In mixture of alkaline phosphatase and vector phage Figure 35c-f, free alkaline phosphatase (dimer size of 94,000
25 daltons) is detected in the flow through as a monomer band with Mr 47,000 on denaturing polyacrulamide gels (figure
35B), while the cross reactive molecule found in vector phage preparations (Mr 45,000) is in retained on the filter (figure
35B). This suggests that the cross reactive molecule is part of the phage particle and underlines the fact that the
ultrafiltration membranes are effecting a separation. Thus the expected fusion band in this phage-enzyme is present
in material retained on ultrafiltration membranes demonstrating that it is part of a larger structure as would be expected
30 for viral bound enzyme.
Catalytic activity has been demonstrated on phage particles expressing alkaline phosphatase. Table 7 shows that
the wild type alkaline phosphatase gene expressed on phage (fd-phoArg1 66) has a specific activity (moles of substrate
converted per mole of viral particles) of 3,700/min. This is close to the turnover value of 4540/min found for purified
alkaline phosphatase by Malamy and Horecker, Biochemistry 3, 1893-1897 1964).
35 Chaidaroglou at al, 1988 supra have shown that substituting alanine for arginine at the active site (residue 166)
leads to a reduction in the rate of catalysis. Preparations of phage displaying alkaline phosphatase with this mutation
derived from TG1 and KS272 show reduced specific activities of 380 and 1400 mol substrate converted/mol phage/
min respectively. Enzyme activity was measured in the retained and flow-through fractions prepared by ultrafiltration,
shown in figure 35. The bulk of activity from phage-enzyme was retained on the filters whereas the majority of activity
40 from free enzyme passes through. Therefore, the enzyme activity in these fusions behaved as would be expected for
virally associated enzyme (not shown). Little or no catalytic activity is measured in preparations of vector phage from
either TG1 or KS272 cells (Table 7), indication that the catalytic activities above are due to phage enzyme and not
contamination with bacterial phosphatase. Addition of phage particles to soluble enzyme does not have a significant
effect on activity (Table 7).
45 Therefore, both the catalytic and immunochemical activity of alkaline phosphatase have been demonstrated to be
due to enzyme which is part of the phage particle.
Example 32
50 Affinity chromatography of phage alkaline phosphatase
Affinity chromatography, using the specific binding properties of enzymes has proved to be a very powerful method
for their purification. The purification of phage-enzymes by this approach would enable the genetic material encoding
the enzyme to be isolated with the enzyme itself. Thus, mutagenesis of cloned enzymes expressed on the surface of
55 filamentous bacteriophage will lead to a whole population of enzyme variants, from which variants with desired binding
properties could be isolated.
Soluble alkaline phosphatase (from calf intestine) has been purified by binding to immobilised arsenate (a com-
petitive inhibitor), and eluting with inorganic phosphate, which is a product (and competitive inhibitor) of the enzyme
54
EP0 774 511 A1
reaction (Brenna,0. at al, Biochem. J. 151 291 -296 1 975). The applicants have determined that soluble alkaline phos-
phatase from E.coli is also retained by this matrix (not shown). In this example it is demonstrated that phage displaying
E.coli alkaline phosphatase binds to arsenate-Sepharose and can be specifically eluted.
Arsenate-Sepharose was prepared by coupling 4-(p-aminophenylazo) phenyl arsonic acid to tyraminyl-Sepharose
5 according to the method of Breena at al, (1975; supra). Affinity chromatography of phage enzyme fdphoArg166 (ex-
ample 31) was carried out in a disposable chromatography column with a 0.5 ml column volume. Columns were pre-
washed with 100 volumes of column buffer (100mM Tris pH 8.4, 1mM MgCI 2 , 0.1 mM ZnCI 2 , 0.1% Tween 20, Brenna
at al, 1975, supra.) 1ml of a 40 fold concentrate of phage-enzyme (in column buffer; prepared as in example 31) was
loaded and washed through with 1 00 volumes of column buffer. Bound phage-enzyme was eluted with 5mls of column
10 buffer containing 20mM NaHP0 4 . The eluate and wash fractions were quantitated by dot blotting onto nitrocellulose
and comparing with known amounts of phage-enzyme. The blots were detected using sheep anti-M1 3 antiserum (gift
from M. Hobart), anti-sheep peroxidase (Sigma) and enhanced chemiluminescent substrate (Amersham). A range of
exposures were taken.
Table 8 shows the results of affinity chromatography of phage displaying alkaline phosphatase on arsenate-Sepha-
15 rose. In separate experiments phage particles expressing either mutant (fdphoAla 166; example 11) and or wild type
(fdphoArg 166) forms are retained on arsenate-Sepharose and eluted with inorganic phosphate. Approximately 0.5 to
3% of added phage enzyme particles loaded ('input phage 1 ) were specifically eluted with phosphate ('output phage')
compared to only 0.05% of vector particles. Arsenate is a competitive inhibitor with Kj of 20jaM with respect to 4-
nitrophenyl phosphate. Phage particles antibodies have previously been isolated on the basis of interactions with similar
20 affinities (example 23). This association is in within the range of a large number of enzyme-ligand interactions sug-
gesting wide applicability for this approach.
Table 8 also shows that the infectivity of phage particles expressing enzyme is reduced with compared with vector
phage particles. This makes titration of infectious particles an inappropriate means of quantitating the number of phage
enzyme particles. For this reason the number of phage were measured by dot blotting and phage were detected with
25 anti-M1 3 antiserum as above.
Whereas, overall recovery of catalytic activity may be an important consideration in enzyme purification, this is not
critical with phage-enzymes. Even if only low levels of phage-enzyme bind to and are specifically eluted from affinity
columns, this will generate clones which can subsequently be grown up in bulk as phage-enzymes or can be transferred
to expression vectors yielding soluble products.
30
Example 33
PCR Assembly of DNA encoding Fab Fragments of an Antibody directed against Qxazolone
35 Example 25 showed that genes encoding Fab fragments could be subcloned into vectors fdCAT2 and pHEN1 and
the protein domains displayed on the surface of phage with retention of binding function. This example shows that the
VHCH and VKCK domains can be amplified separately and then joined by a linker allowing the expression of the light
chain as a genelll protein fusion and the VHCH fragment as a soluble molecule. A functional Fab fragment is then
displayed on phage by association of these domains. The assembly process, described in this example, is required
40 for display of a library of Fab fragments derived from the immune repertoire if both heavy and light chain domains are
to be encoded within a single vector.
The VHCH1 and VKCK domains of- a construct (example 25; construct II in pUC1 9) derived from antibody NQ10
12.5 directed against 2-phenyl-5-oxazolone were amplified using PCR. For cloning into the vector fdCAT2 the oligo-
nucleotides VH1 BACKAPA (example 25) and HulgG1-4 CH1 FOR (example 40) were used to amplify the VHCH1 do-
45 mains. For cloning into pHEN1 VH1BACKSFH5 (example 25) replaced VH1 BACKAPA for this amplification. For cloning
into both vectors the VKCK domains were amplified using VK2BACK (example 25) and CKNOTFOR (example 40). A
linker oligonucleotide fragment containing the bacteriophage fd gene 8 terminator and the fd gene 3 promoter was
prepared by amplifying the region containing them from the vector fdCAT2 by PCR using the oligonucleotides.
50
VK- TERM -FOR
5* TGG AGA CTG GGT GAG CTC AAT GTC GGA GTG AGA ATA GAA
AGG 3' (overlapping with VK2BACK [example 141V
55 and
55
EP0 774 511 A1
CHI -TERM-BACK
5 1 AAG CCC AGC AAC ACC AAG GTG GAC AAG AAA GTT GAG CCC AAA
TCT AGC TGA TAA ACC GAT ACA ATT AAA GGC 3 1 (overlapping
5 with HuIgGl-4 CH1-FOR)
Assembly of the Fab fragment from the amplified VHCH1 and VKCK domains and the linker prepared as above was
as described in example 14E except that the primers VH1 BACKAPA (when cloning into fdCAT2) or VH1 BACKSFH5
(when cloning into pHEN1) and CKNOTFOR were used for the final reamplification, thereby introducing restriction
10 sites for cloning into fdCAT2 (Apall-Notl) or pHEN1 (Sfil-Notl) the assembled Fab fragment is shown in figure 34. No
assembled product was seen in the absence of linker. An assembled scFv prepared according to example 14 is shown
for comparison.
Phage antibodies were prepared as in example 25 and ELISA was performed with oxazolone as antigen according
to example 6. Results were as expected for Fab fragments cloned in both fdCAT2 and pHEN1 samples, phage particles
15 bound to oxazolone as detected by a positive ELISA signal.
Example 34
Construction of a Gene III Deficient Helper Phage
20
To fully realise the potential of the phagemid cloning system, a helper phage lacking gene III is desirable. Rescue
of gene III fusions with such a helper phage would result in all the progeny phagemids having a gene III fusion on their
capsid, since there would be no competition with the wild type molecule.
Control over the number of fusion molecules contained on each phage will provide particularly useful. For example,
25 a gene III deficient helper phage can be used to rescue low affinity antibodies from a naive repertoire, in which high
avidity will be necessary to isolate those phage bearing the correct antibody specificity. The unmutated helper phage
can then be used when higher affinity versions are constructed, thereby reducing the avidity component, and permitting
selection purely on the basis of affinity. This will prove a surprisingly successful strategy for isolation and affinity mat-
uration of antibodies from naive libraries.
30 The strategy chosen to construct the helper phage was to partially delete gene III of Ml 3K07 using exonuclease
Bal 31 . However, phage lacking gene III protein are non-infective so an E.coli strain expressing gene III was constructed.
Wild type M13 gene III was PCR-amplified with primers glllFUFO and glllFUBA, exactly as described in example 24.
The PCR product was digested with Eco Rl and Hind III and inserted into Eco Rl and Hind Ill-cut pUC1 9 (not a phagemid
as it lacks the filamentous phage origin of SS DNA replication) under control of the lac promoter. The plasmid was
35 transformed into E.coli TG1 , and the resulting strain called TG1/pUC1 9glll. This strain provides gill protein in trans to
the helper phage.
There is a single unique Bam HI site in M13K07, which is approximatlely in the centre of gill. Doublestranded
M13K07 DNA was prepared by alkaline lysis and caesium chloride centrifugation (Sambrook at al, at supra. 1989);
twenty u.g of DNA was cut with Bam H1 , phenol extracted and ethanol precipitated then resuspended in 50uJ of Bal 31
40 buffer (600mM NaCI, 20mM Tris-HCI pH 8.0, 12 mM CaCI 2 , 1 2mM MgCI 2 and 1 mM EDTA) and digested for 4 minutes
with 1 unit of Bal 31 (New England BioLabs). This treatment removed approximatley 1 Kb of DNA. EGTA was added
to 20mM and the reaction phenol extracted and ethanol precipitated prior to purification of the truncated genome on
an agarose gel. The DNA was repaired with klenow enzyme and self-ligated with T4 DNA ligase (New England Biolabs).
Aliquots of the ligation reaction were transformed into competent TG1/pUC1 9glll and plated on SOB medium
45 containing ampicillin at 100|ag/ml and kanamycin at 50u.g/ml. Colonies were screened for the presence of a deletion
by PCR with primers glllFUBA and KSJ12 (CGGAATACCCAAAAGAACTGG).
KSJ 12 anneals to gene VI which is immediately downstream of gill in the phage genome, so distinguishing gill
on the helper phage from that resident on the plasmid. Three clones gave tructated PCR products corresponding to
deletions of ca. 200, 400 and 800bp. These clones were called M1 3K07 gill A Nos 1 ,2 and 3 respectively. No clones
50 were isolated from the earlier Bal 31 time points, suggesting that these are in some way lethal to the host cell. Several
clones were isolated from later time points, but none of these gave a PCR product, indicating that the deletion reaction
had gone too far.
M1 3K07 .gill A No.s 1 ,2 and 3 were cultured and the resulting helper phage tested for their ability to rescue an
antibody gill fusion (scFv D1 .3) by ELISA, exactly as described in example 18. As shown in figure 37, only one clone,
55 M13K07 gill A No3 was found to rescue the antibody well; in fact the signal using this helper was greater than that
observed with the parent M13 K07. M13K07 glllA No3 rescued phagemids should have a much higher density of
antibody fusions on their surfaces. That this was indeed the case was demonstrated when the phage used in this
ELISA were analysed by Western blotting with anti gill protein antiserum (fig. 38). This analysis enables estimation of
56
EP0 774 511 A1
the amount of gill fusion protein versus free gill protein present on the phage(mid) particles.
Only a minute fraction of the gill protein on the M1 3K07- rescued material is present as an intact fusion (fig 38).
The fusion protein band is induced by IPTG, so is indisputably that synthesised by the phagemid. As expected, even
when the lac promoter driving gill fusion protein synthesis is fully induced (100uJvl IPTG), wild type gill protein, at a
lower copy number and driven from a far weaker promoter, predominates. This is in contrast to the pattern generated
by the same clone rescued with M1 3K07 .glllANo3, and the pattern generated by fd CAT2-scFv D1 .3. In both of these
latter cases, there is no competition with wild-type gill and the fusion protein band is correspondingly stronger.
It is worthy of note that construction of M13K07 gill A No3 was immensely inefficient: one clone from 20uxj of
starting DNA. Moreover, the yield of gill helper phage from overnight cultures is extremely low ca.10 6 cfu/ml compared
with ca. 10 11 cfu/ml for the parental phage. Despite this, M1 3K07 gill No3 rescues the phagemid as well as the parental
phage, as judged by the number of phagemid particles produced after overnight growth. This indicates that trans
replication and packaging functions of the helper are intact and suggest that its own replication is defective. Hence it
may be that inactivation of gill is normally toxic to the host cell, and that M13K07 gill A No3 was isolated because of
a compensating mutation affecting, for example, replication. Phage fd-tet is unusual in that it tolerates mutations in
structural genes that are normally lethal to the host cell, since it has a replication defect that slows down accumulation
of toxic phage products; M1 3K07 gill A No3 may also have such a defect.
M1 3K07g III A No 3 has been deposited at the National Collection of Type Cultures, 61 Colindale Avenue, London,
NW9 6HT, UK (Accession No. NCTC 12478). On 28 June 1991, in accordance with the regulations of the Budapest
Treaty. It contains a deletion of the M13 genome from bases 1979 to 2768 inclusive (see Van Wezenbeek, P.G.M.F.
at al., Gene II p129-148, 1980 for the DNA sequence of the M1 3 genome).
Example 35
Selection of bacteriophage expressing scFv fragments directed against lysozyme from mixtures according to affinity
using a panning procedure
For isolation of an antibody with a desired high affinity, it is necessary to be able to select an antibody with only a
few fold higher affinity than the remainder of the population. This will be particularly important when an antibody with
insufficient affinity has been isolated, for example, from a repertoire derived from an immunised animal, and random
mutagenesis is used to prepare derivatives with potentially increased affinity. In this example, mixtures of phage ex-
pressing antibodies of different affinities directed against hen egg lysozyme were subjected to a panning procedure.
It is demonstrated that phage antibodies give the ability to select for an antibody with a K d of 2nM against one with a
K d of 13nM.
The oligonucleotides used in this example are shown in the list below:
OLIGONUCLEOTIDES
VHBHD13APA :
: 5 ' -
CAC
AGT
GCA
CAG
GTC
CAA
CTG
CAG GAG AGC
GGT
VHFHD13 :
: 5 ' -
CGG
TGA
CGA
GGC
TGC
CTT
GAC
CCC
HD13BLIN :
: 5 1 -
GGG
GTC
AGG
GCA
GCC
TCG
TCA
CCG
HD13FLIN3 :
: 5 ' -
TGG
GCT
CTG
GGT
CAT
CTG
GAT
GTC CGA T
VKBHD13 :
; 5 ' -
GAC
ATC
CAG
ATG
ACC
CAG
AGC
CCA
VKFHD13NOT
CAC CTT GGT
MURD13SEQ
HUMD13SEQ
FDPCRFOR
FDPCRBAK
5 ' -
CCC
5 1 -
5 ' -
5 1 -
5 ' -
GAG TCA TTC TGC GGC CGC ACG TTT GAT TTC
GAG GAG ATT TTC CCT GT
TTG GAG CCT TAC CTG GC
TAG CCC CCT TAT TAG CGT TTG CCA
GCG ATG GGT GTT GTC ATT GTC GGC
Phage displaying scFv fragments directed against lysozyme were derived from cloned Fab fragments in plasmids.
Heavy and light chain variable regions were amplified by the polymerase chain reaction (PCR) from plasmids
containing humanized VH-CH1 or VK-CK inserts suitable for production of Fab fragments (gift of J. Foote). The disso-
ciation constant, Kd for different combinations of the two plasmids combined as Fabs, are shown below:
57
EP0 774 511 A1
Heavy Chain Plasmid
Light Chain Plasmid
Kd
HuH-1
HuK-3
52 nM
HuH-1
HuK-4
180 nM
HuH-2
HuK-3
13 nM
HuH-2
HuK-4
(not determined)
Primary PCR
w
The primary PCR of the variable regions was performed by combining the following:
36.5 jlxI Water
5 jlxI PCR buffer (10x)
-is 2 jlxI dNTP (5mM)
2.5 uJ Backoligo (10 pmoles/uJ (VHBHD13APA or VKBHD13)
2.5 uJ Forward oligo (10 pmoles/uJ (VHFHD13 or VKFHD13NOT)
The reaction is decontaminated by UV irradiation to destroy foreign DNAfor 5 minutes, and 1 uJ of plasmid DNA
20 added (0.1 uxj/ul). The per mixture was covered with 2 drops of paraffin oil, and placed on the per block at 94°C for 5
minutes before the addition of 0.5 uJ of Tag DNA polymerase under the paraffin. The cycling conditions used were
94°C 1 min, 40°C 1 min, 72°G 1 .5 min 1 7 cycles.
The linker (Gly 4 -Ser) 3 , was amplified from the anti-phOx (2-phenyloxazol-5-one) clone fd-CAT2-scFv NQ11 , using
the oligos HD13BLIN and HD13FLIN3, with 0.1 ng of plasmid DNA. The PCR cycling used was 94°C 1 min, 25°C 1.5
25 min, for 17 cycles.
Amplified DNA was purified by running the samples on a 2% low melting point agarose gel at 90 mA, excising the
appropriate bands and extracting the DNA using the Geneclean II Kit (BIO 101 Inc.) for the VH and VK, or by using
Spin-X filter units (Costar) for the linker. A final volume of 1 0 jlxI was used to resuspend the extracted DNA.
30 PCR Assembly
Assembly of the four single chain Fv Humanized D1 .3 (scFv HuD1 .3) constructs was by the process of 'assembly
by overlap extension' example 14.
The following were combined:
35
34.5 uJ Water
5 uJ PCR Buffer (10x)
2 uJ dNTP (5 mM)
2.5 uJ Backoligo (10 pmoles/uJ) (VHBHD1 3APA)
40 2.5 jllI Forward oligo (10 pmoles/uJ) (VKFHD1 3NOT)
Once again, the reaction is decontaminate by UV treatment for 5 minutes before the addition of 1 jlxI of the primary
PCR products; VH-1 or VH-2, VK-3 or VK-4, plus the linker DNA. The reaction was covered with 2 drops of paraffin,
and heated at 94°C for 5 minutes before the addition of 0.5 uJ of Tag Polymerase. The PCR cycling conditions used
were 94°C 1 min, 60°C 1 .5 min, 72°C 2.5 min for 20 cycles.
The aqueous layer under the paraffin was extracted once with phenol, once with phenol: chloroform, once with
ether, ethanol precipitated, and resuspended in 36 jlxI of water. To this was added, 5 uJ of 10x Buffer for Notl, 5 uJ 1 mg/
ml BSA, and 4 uJ (40 U) of Notl (New England Biolabs). The restriction was incubated at 37°C overnight.
The DNA was ethanol precipitated and resuspended in 36 uJ of water, and 5 jlxI 10x NEB Buffer 4, 5 uJ 1 mg/ml
BSA, and 2 jllI (40 U) of ApaLI (New England Biolabs). This was incubated at 37°C for 5 hours; a further 2 uJ of ApaLI
was added and the reaction incubated at 37°C overnight.
The cut DNA was extracted by gel purification on a 1 .3% low melting point agarose gel followed by treatment with
Geneclean, to yield the insert DNA for cloning.
Vector fd CAT2 (prepared and digested with ApaLI and Notl as in example 20) and the scFv DNA were ligated as
in example 20.
58
EP0 774 511 A1
Analysis Of Clones
Colonies from the ligations were first screened for inserts by PCR screening. The PCR mixture was prepared in
bulk by combining 14.8 uL 1x PCR Buffer, 1 uJ dNTP (5 mM), 1 jlxI Back oligo (FDPCRBAK), 1 jlxI Forward oligo (FD-
5 PCRFOR), and 0:2 uJ Tag polymerase per colony screened. 20 uJ of this PCR mixture was aliquoted into a 96 well
Techne plate. The top of a colony was touched with a toothpick and twirled quickly into the PCR mixture and the colony
rescued by placing the toothpick in a Cellwell plate (Nunc) containing 250 uJ of 2x TY medium. The PCR mixture is
covered with 1 drop of paraffin and the plate placed on the block at 94°C. for 10 minutes before cycling at 94°C 1
minute, 60°C 1 minute, 72°C 2.5 minutes.
10 The clones thus derived were named as below. The affinity of scFv fragments derived the Fab fragments was not
determined but previous results suggests that these are closely related although not necessarily identical (R.E. Bird
& B.W. Walker TIBTECH 9 132-137, 1991).
Construct Name
Composition
Affinity of Fab(Kd)
TPB1
VH-HuH2-(Gly 4 -Ser) 3 -VK-HuK3
13 nM
TPB2
VH-HuH1-(Gly 4 -Ser) 3 -VK-HuK4
180 nM
TPB3
VH-HuH2-(Gly 4 -Ser) 3 -VK-HuK4
(Unknown)
TPB4
VH-HuH1-(Gly 4 -Ser) 3 -VK-HuK3
52 nM
20
Preparation of phage and ELISA was as described in example 6. The clones generated in fd CAT2 were shown
to bind lysozyme as expected.
Affinity selection
25
Selection of Highest Affinity Binding Phage
Mixing experiments were performed in which fd-CAT2 scFvD1.3 phage (example 19) were mixed with either fd-
CAT2 TPB1 , fd-CAT2 TPB2, or fd-CAT2 TKPB4, and used in one round of panning.
The general method used for affinity selection by panning is that detailed below. Any deviation from this protocol
is described at the relevant point. Panning plates were placed on a rocking platform between manipulations.
Falcon 35 mm Tissue Culture dishes were coated overnight with 1 ml of Lysozyme (various concentrations) dis-
solved in 50 mM Sodium Hydrogen Carbonate, pH 9.6, and blocked with 2 ml 2% MPBS at room temperature for 2
hours. Phage were prepared in 1 ml 2% MPBS and rocked at room temperature for 2 hours. Plates were washed for
5 minutes with 2 ml of the following solutions; 5 times with PBS, PBS-Tween, 50 mM Tris-HCI, pH 7.5; 500 mM Sodium
Chloride, 50 mM Tris-HCI, pH 8.5; 500 mM Sodium Chloride, 50 mM Tris-HO, pH 9.5; 500 mM Sodium Chloride, 50
mM Sodium Hydrogen Carbonated, pH 9.6; 500 mM Sodium Chloride. Phage were then eluted by adding 1 ml 100
mM Triethylamine and rocking for 5 minutes before removing the eluate which was neutralised with 1 00 uJ 1 .0 M Tris-
HCI, pH 7.4.
Plates were coated overnight with Lysozyme at the concentration listed below.
Colonies from the single round of panning were probed with either MURDSEQ (for fdCAT2 scFvD1.3) or
HUMD1 3SEQ (for fdCAT2 TPB constructs).
Circles of nitrocellulose (Schleicher & Schuell, BA 85, 0.45 jam) were labelled in pencil and lowered gently onto
the colonies derived from the panning experiments and left for one minute. The filters were then pulled off quickly from
one edge and placed colony side up on a piece of 3MM paper (Whatman) soaked in Denaturing solution (500 mM
Sodium Hydroxide; 1 .5 M Sodium Chloride) for 5 minutes. They were then transferred to 3MM soaked in Neutralizing
Solution (3.0 M Sodium Chloride; 500 mM Tris-HCI, pH 7.5) for 1 minute, and then to 3MM soaked in 5x SSC; 250 mM
Ammonium Acetate for 1 minute. The filters were then air dried before baking in an 80°C vacuum oven for 30 minutes.
The oligonucleotide probe was prepared by combining the following:
2 uJ oligonucleotide (1 pmoles/|Lil)
2 uJ y-32P ATP (3000 Ci/mmole) (Amersham International pic)
2 |llI 10 x Kinase buffer (0.5 M Tris-HCI, pH 7.5; 100 mM Magnesium Chloride; 10 mM DTT)
12 uJ Water
2 jlxI Polynucleotide Kinase (20 Units)
This was incubated at 37°C for 1 hour.
Hybridization was performed in the Techne HB-1 Hybridiser. The baked filters were pre-hybridized at 37°C in 40
59
EP0 774 511 A1
ml of Hybridization Buffer (10 ml 100 mM Sodium pyrophosphate; 180 ml 5.0 M Sodium chloride; 20 ml 50x Denharts
Solution; 90 ml 1.0 M Tris-HC1, pH 7.5; 24 ml 250 mM EDTA; 50 ml 10% NP40; made to 1 litre with water; 60.3 mg
rATP; 200 mg yeast RNA (Sigma)), for 1 5 minutes before the addition of the 20 jlxI of the kinased oligo. The filters were
incubated at 37°C for at least one hour, and then washed 3 times with 50 ml of 6x SSC at 37°C for 10 minutes (low
s stringency wash). Filters were air dried, covered with Saran wrap and exposed overnight with Kodak X-AR film.
Selection of fd-CAT2 scFv D1.3 from fd-CAT2 TPB4
Figure 39, summarizes the results from panning experiments using a mixture of the high affinity fd-CAT2 scFv
10 D1 .3 phage (Kd-2 nM) and the fd-CAT2 TPB4 construct (Kd-52 nM).
At a coating concentration of 3000 jag/ml Lysozyme, little or no enrichment could be obtained. It was however,
possible to get enrichment for the scFv D1 .3 phage when a lower concentration of Lysozyme was used for coating the
plates. The best enrichment value obtained was from 1 .5% fd-CAT2 scFv D1 .3 in the starting mixture, to 33% fd-CAT2
scFv D1 .3 in the eluted faction, on a plate coated overnight with 30 |LLg/ml Lysozyme.
15
Selection of fd-CAT2 scFv D1.3 from fd-CAT2 TPB1
Enrichment for the high affinity scFv D1 .3 phage over the fd-CAT2 TPB1 phage (Kd-1 3) nM, could only be shown
from experiments where the plates had been coated overnight with low concentrations of Lysozyme, as shown in Figure
20 40.
In summary, single chain Fv versions of a series of humanized D1 .3 antibodies have been constructed in phage
fd-CAT2. By affinity selection of fd-CAT2 phage mixtures, by panning in small petri dishes, it was shown that the high
affinity scFv D1 .3 phage, could be preferentially selected for against a background of lower affinity scFv HuD1 .3 phage.
25 Example 36
Expression of Catalvtically Active Staphylococcal Nuclease on the Surface of Bacteriophage fd
Examples 11 and 12 showed that alkaline phosphatase from E.coli can be expressed as a catalvtically active
30 enzyme on the surface of bacteriophage fd. Here we show that Staphylococcal nuclease can also be expressed in a
catalvtically active form suggesting that this methodology may be general.
The gene for the enzyme Staphylococcal nuclease (SNase) was amplified from M13 mp18 - SNase (Neuberger,
M.S. at a] Nature 312 604-608, 1984) by PCR using primers with internal ApaLI (5'-GGAATTCGTGCACAGAGT-
GCAACTTCAACTAAAAAATTAC-3') and Notl (5'-GGGATCCGCGGCCGCTTGACCTGAATCAGCGTTGTCTTCG-3')
35 restriction sites, cloned into phage vector fd-CAT2 after digestion with ApaLI -Notl restriction enzymes and the nucle-
otide sequence of the SNase gene and junctions with gene III checked by DNA sequencing. The fd-tet-SNase phage
was prepared from the supernatant of infected E.coli TG1 cultures by three-rounds of PEG precipitation, and the fusion
protein demonstrated by SDS-gel electrophoresis and Western blotting using rabbit anti-g3p antiserum (Prof. I. Ra-
sched, Konstanz) and peroxidase-labelled goat anti-rabbit antibodies (Sigma) (Fig. 41 ) as described in example 27. As
40 well as the fusion protein band (calculated Mr 59749, but runs at a higher position due to the aberrant g3p behaviour),
a smaller (proteolytic ?) product is seen.
The fusion protein was shown to be catalvtically active by incubation of the fd-tet-SNase phage (4 x 10 9 tetracyclin
resistant colonies [TU]) with single stranded DNA (1 jLxg) for 1 hr at 37°C in the presence of Ca 2 +, and analysis of the
digest by agarose gel electrophoresis (Figure 42). Nuclease activity was not detected with the parent fd-CAT2 (2 x
45 10 10 TU) phage alone or after three rounds of PEG precipitation of mixtures of fd-CAT2 (2 x 10 10 TU) with SNase (0.7
jug). Thus the nuclease activity results from the display of the enzyme on the surface of the phage and not from co-
precipitated or soluble SNase set free by degradation of the fusion protein. The nuclease activity of fd-tet-SNase (Figure
42) lies in the same order of magnitude, (2 x 10 8 TU and assuming three copies of SNase per TU) as an equimolar
amount of SNase (0.03 ng or 10 9 particles), and like the authentic SNase was dependent on Ca 2 +, since incubation
50 with 40 mM MgCI 2 and 25 mM EGTA blocked activity (not shown).
Example 37: Display of the Two Aminoterminal Domains of Human CD4 on the Surface of fd Phage
The protein CD4, a member of the immunoglobulin superfamily, is a cell surface receptor involved in MHC class
55 || restricted immune recognition. It is also recognised by the protein gp120 derived from the human immunodeficiency
virus (AIDS virus). The first two domains (named V1 and V2, residues 1-178) of the surface antigen CD4 were amplified
from pUC1 3-T4 (gift from T. Simon) containing the human cDNA of CD4, by PCR using primers with internal ApaLI (5'-
GGA ATT CGTGCACAG AAG AAA GTG GTG CTG GGC AAA AAA GGG G-3') and Notl (5'-GGG ATC CGC GGC
60
EP0 774 511 A1
CGC AGC TAG CAC CAC GAT GTC TAT TTT GAA CTC-3') restriction sites. After digestion with these two enzymes,
the PCR-product was cloned into fdCAT2, and the complete nucleotide sequence of the CD4-V1 V2 DNA and junctions
with gene III checked by dideoxy sequencing using oligonucleotides fd-seql (5'-GAATTT TCT GTATGAGG), CD4-seql
(5'-GAA GTT TCC TTG GTC CC-3') and CD4-seq2 (5'-ACT ACC AGG GGG GCT CT-3'). In the same way, a fd-CD4-V1
5 version was made, linking residues 1-107 to the N-terminus of gene III, using previously mentioned primers and oli-
gonucleotide 5'-GGG ATC CGC GGC CGC GGT GTC AGA GTT GGC AGT CAA TCC GAA CAC-3' for amplification,
PCR conditions and cloning were essentially as described in example 15 except that digestion was with ApaLI and
Notl (used according to the manufacturers instructions).
Both fd-CD4-VI and fd-CD4-VIV2 phages were prepared from the supernatant of infected E.coli TG1 cultures by
10 three rounds of PEG precipitation, thereby concentrating the sample 100-fold for ELISA analysis. The fusion protein
was detected in a Western blot (results not shown) with a rabbit anti-gene III antiserum, and revealed bands of the
expected size.
Binding of the CD4 moiety to soluble gp120 (recombinant HIV-IIIB gp120 from CHO cells, ADP604, obtained from
the Aids Directed Programme, National Institute for Biological Standards and Controls, South Mimms, Potters Bar, UK)
15 was analysed in an ELISA, using 5 jag/ml gp120 for coating (overnight, in PBS). Anti-M1 3 antiserum was used to detect
bound phage; all other conditions were as in Example 9. Figure 43 shows the ELISA signals of wild-type phage (fd-
tet) and both CD4-phages. Both CD4-phages can bind gp120, but fd-CD4-V1 V2 binds much stronger to gp120 than
fd-CD4-V1. The binding competitors, soluble CD4 (recombinant soluble CD4 from Baculovirus, ADP 608; from the
AIDS Directed Programme) (25 |ug/ml) or soluble gp1 20 (20 |ug/ml), added together with the 50 jlxI phage stock sample
20 during the ELISA, decreased the signal to background level. These results indicate that phage binding to gp120 is
mediated by the CD4 molecule displayed at its surface,, and that binding is stronger when the two aminoterminal
domains of CD4 are presented.
Thus, CD4 is a cell surface receptor molecule which is active when displayed on bacteriophage fd. Like the PDGF-
BB receptor, thefunctional display of which is described in examples 15 and 16, CD4 is a memberof the immunoglobulin
25 superfamily and this result suggests that this class of molecule may be generally suitable for display on the surface of
phage.
Example 38 Generation and Selection of Mutants of an Anti-4-hydroxv-3-nitrophenylacetic acid (NP) Antibody
expressed on Phage using Mutator strains
30
It will sometimes be desirable to increase the diversity of a pool of genes cloned in phage, for example a pool of
antibody genes, or to produce a large number of variants of a single cloned gene. There are many suitable in vitro
mutagenesis methods. However, an attractive method, particularly for making a more diverse population of a library
of antibody genes, is to use mutator strains. This has the advantage of generating very large numbers of mutants,
35 essentially limited only by the number of phage that can be handled. The phage display system allows full advantage
to be taken of this number to isolate improved or altered clones.
Nucleotide sequences encoding an antibody scFv fragment directed against 4-hydroxy-3-nitrophenylacetic acid
(NP), scFvBI 8, derived as in example 1 4 from a monoclonal antibody against NP were cloned into fdCAT2 using ApaLI
and Notl restriction sites as in example 11 to create fdCAT2scFvB18 or into fdDOGKan (fdCAT2 with its tetracycline
40 resistance gene removed and replaced by a kanamycin resistance gene) using Pstl and Notl restriction sites to create
fdDOGKanscFvBI 8 or into the phagemid vector pHEN1 using the restriction sites sfil and Notl as a fusion protein with
gene III to create pHEN1scFvB18.
The following mutator strains (R. M. Schaaper & R.L. Dunn J. Mol. Biol. 262 1627-16270, 1987; R. M. Schaaper
Proc. Natl. Acad. Sci. U.S.A. 85 8126-8130 1988) were used:
45
NR9232: are, thi, mutD5-zaf1 3::Tn10, prolac, F'prolac
NR9670: are, thi, azi, mutTI, leu::TnlO, prolac
NR9292: are, thi, mutH101, prolac, F'prolac
NR9084: are, thi, mutTI, azi, prolac, F'prolac|-Z-AM15 M15
50 NR9046: are, thi, supE, rif, nalA, metB, argE(am), prolac, F'prolac
were kind gifts of Dr. R. M. Schaaper (Department of Health & Human Services, N1H, PO Box 12233, Research
Triangle Park, N.C. 27709)
NR9046mutD5: NR9046 mutD5::Tn10
NR9046mutT1: NR9046 mutTI::Tn10
55
were constructed by P1 transduction according to standard procedures. Mutator strains were transfected with
fdCAT2scFvB18 of fdDOGKanscFvBI 8 and transfectants selected for antibiotic resistance. Transfectants were grown
for 24h at 37°C before mutant phage was harvested by PEG precipitation. The mutant phage were selected on a 1ml
61
EP0 774 511 A1
NIP (4-hydroxy-3-iodo-5nitrophenylacetic acid)-BSA-Sepharose affinity column (prepared according to the manufac-
turers instructions) prewashed with 200ml of PBS and blocked by 20ml MPBS. Phage were loaded on the column in
10ml MPBS and unbound material reapplied to ensure complete binding. The column was subsequently washed with
10ml of MPBS and 500ml of PBS. Phage bound to the affinity matrix was eluted with 5 column volumes of 0.33 mM
5 NIP-Cap (example 48).
Phage eluate was incubated for 30min to 1 h with log phase (2x1 0 8 cells/ml) E.coli mutator strains without antibiotic
selection. The infected cells were then diluted 1:100 in 2xTY and grown for 24h with antibiotic selection (15jag/ml
tetracyclin or 30|ag/ml kanamycin for fdCAT2scFvB18 or fdDOGKanscFvB18 respectively). Phage from this culture
was used for another round of affinity selection and mutation.
10 Binding of phage antibodies was assayed by ELISA as in example 9 except that ELISA plates were coated with
NIP-BSA (4-hydroxy-3-iodo-5-nitrophenylacetyl-BSA; 0.4 mg/ml). Culture supernatants were prepared following
growth in Cellwells as described in example 21 and 20uJ of culture supernatant was added to each well diluted to 200uJ
with MPBS.
Phage samples giving signals in ELISA of more than twice the background were tested ELISA as above for non-
75 specific binding against lysozyme, BSA or Ox-BSA (example 9). Specificity for NIP was further confirmed by an ELISA
in which serial dilutions of NIP-CAP were added together with phage antibodies. Addition of increasing concentrations
of NIP-CAP reduced the ELISA signal to the background level.
Phage giving positive signals in ELISA were sequenced and 2 different mutants were subcloned into pHEN1
phagemid and transformed into HB2151 for soluble expression and TG1 for phage display (example 27).
20 For expression of soluble scFv fragments, transformants in E.coli HB2151 were grown at 37°C in 1 litre 2xTY,
0.2% glucoe, 0. 1 mg/ml ampicillin to an OD600 of 1 and expression of soluble scFv fragments induced by adding I PTG
to 1mM. Cultures were shaken at 30°C for 16h.
Soluble scFvB18 was concentrated from crude bacterial supernatant in a FLOWGEN ultrafiltration unit to a volume
of 200ml.
25 The concentrate was passed two times over a 2ml column of NIP-BSA-Sepharose prewashed with 200ml of PBS.
The column was washed with 500ml of PBS and 200ml of 0.1 M Tris pH7.5, 0.5M NaCI and phage antibodies eluted
with 50mM Citrate buffer pH2.3. The eluate was immediately neutralised with IMTris pH8. The eluate was dialysed
against two changes of 1 litre PBS, 0.2mM EDTA, Precipitated protein was removed by centrifugation at 10000g and
protein yield was determined by measuring the absorbance at 280nm of the supernatant.
30 After 4 rounds of mutation and selection, isolated clones were screened and in one or two rare examples strongly
positive ELISA signals were obtained from phage antibodies derived from the mutation of each of fdCAT2scFvB1 8 and
fdDOGKanscFvB18 in the ELISA. The ELISA conditions were such that the parent phage fdCAT2scFvB18 only gen-
erated weak signals. These phage antibodies giving strongly positive ELISA signals were enriched in further rounds
by a factor of roughly 2.5 per round. Forty phage antibodies giving strongly positive signals were sequenced and they
35 each displayed single mutations in six different positions in the scFvB18 nucleotide sequences, five of which reside in
the light chain. More than 70% of the mutations occurred at positions 724 and 725 changing the first glycine in the J
segment of the light chain (framework 4) to serine (in 21 cases) or aspartate (in 3 cases). The mutations found are
shown in Table 9. The sequence of scFvB18 is shown in Figure 44.
The nucleotide sequences encoding the scFv fragments of a framework mutant with the above glycine to serine
40 mutation, as well as a mutant where Tyr in the CDR3 of the light chain had been mutated to aspartate, were amplified
by PCR from the phage antibody clones and subcloned into pHEN1 phagemid (essentially as in example 25). This
avoids possible problems with genelll mutations caused by the mutator strains. The same pattern of ELISA signals
was seen when the mutants were displayed on phage following rescue of the phagemid with helper phage (as described
in example 25) as when the mutants were assayed when expressed from the phage genome as above.
45 The scFv fragments from scFvBI 8 and the scFv fragments containing the glycine to serine and tyrosine to aspartate
mutations respectively were expressed in solution (following transformation into E.coli HB2151 as in example 27) at
30°C. They showed no differences in the ELISA signals between wild-type B1 8 and the framework mutant. The signal
obtained from the phage antibody with the Tyr mutated to aspartate in CDR3 of scFvB18 was about 10x stronger.
Expression yields were found to be comparable as judged by Western blotting using an antiserum raised against g3p
50 (as described above). Affinity measurements were performed using fluorescence quenching as described in example
23. Affinity measurement of affinity purified scFv fragments however showed scFvB18, and the scFvB18 (Gly->Ser)
and scFvB18(Tyr->Asp) mutants all to have a comparable affinity of 20nM for NIP-CAP.
A Western blot using an anti-genelll antibody showed the framework mutant had suffered significantly less prote-
olytic cleavage than scFvB18.
55 Hence, the use of mutator strains generates a diverse range of mutants in phage antibodies when they are used
as hosts for clones for gene III fusions. In this case some of the clones exhibit higher ELISA signals probably due to
increased stability to proteolyic attack. The mutator strains can therefore be used to introduce diversity into a clone or
population of clones. This diversity should generate clones with desirable characteristics such as a higher affinity or
62
EP0 774 511 A1
specificity. Such clones may then be selected following display of the proteins on phage.
Example 39 Expression of a Fv Fragment on the Surface of Bacteriophage by Non-Covaient Association of VH and
VL domains
5
This example shows that functional Fv fragments can be expressed on the surface of bacteriophage by non-
covalent association of VH and VL domains. One chain is expressed as a gene III fusion and the other as a soluble
polypeptide. Thus Fv fragments can be used for all the strategies discussed for Fab fragments including dual combi-
natorial libraries (example 26).
10 A useful genetic selection system for stably associated Fv fragments could be established if the expression of Fv
fragments as fusion proteins on the phage surface would be possible such that one V domain is fused to the gene III
protein and the other Vdomain is expressed separately in secreted form, allowing it to associate with the V domain on
the fusion protein provided the interaction strength is sufficiently high. This idea was tested in a model experiment
using the Vdomains from the anti-hen egg lysozyme antibody D1 .3 by fusing the D1 .3 VK gene to gene III and separately
15 expressing the D1 .3 VH domain.
Experimentally this was achieved as follows: The vector fd-DOG1 was digested with the restriction enzymes Pstl
and Xhol. From the Fv expression plasmid pSW1-"VHD1 .3-VKD1 .3myc version 3/pUC119 (Ward et al., 1989 supra)
a Pst 1/Xho I -digested restriction fragment was isolated that carries the VH domain coding sequence (terminated by
2 stop codons), a spacer region between VH and VK genes including a ribosome-binding site for expression of the VK
20 gene, a pelB leader sequence, and, following in frame, the VK gene. This fragment was cloned into the digested fd-
DOG vector to generate the construct fd-tet Fv D1 .3. As shown on the map in Fig. 45, the dicistronic VH/VK-gene III
operon is transcribed from the gene III promoter; secretion of the VH domain is achieved by the gene III protein leader,
secretion of the VK-genelll fusion protein by the pelB leader sequence. For control purposes a second construct with
the name fd-tet Fv D1.3 (AS-Stuffer) was made by a similar route as described above: the VH used in this construct
25 carries an insertion of a 200 bp fragment in the Sty I restriction site at the junction of VH CDR 3/FR4, thus interrupting
the VH with several in frame stop codons. It is known from previous work that this insertion sufficiently disrupts the VH
structure to abolish binding to the antigen lysozyme when expressed either as a soluble Fv or single-chain Fv fragment
or as a single-chain Fv fragment on phage surface. This construct was used as a control. TG1 bacteria carrying either
the fd-tet Fv D1 .3, fd-tet Fv D1 .3 (AS-Stuffer) or as single-chain wild-type control fd-tet scFv D1 .3 plasmids were grown
30 in liquid culture (medium 2xTY containing 1 5 |ug/ml tetracycline) for 24h to produce phage particles in the supernatant.
After removal of bacterial cells by centrifugation the phage titer in the supernatants was determined by re-infecting
exponentially growing TG1 cells with dilutions of the supernatants and scoring tetracycline-resistants colonies after
plating on tetracycline-plates. The infectious phage titers achieved were 1x1 0 11 tetR transducing units/ml for the single-
chain wild-type control fd-tet scFv D1.3 and 2x1 0 10 tetR transducing units/ml for Fv phage constructs fd-tet Fv D1.3
35 and fd-tet Fv D1 .3 (AS-Stuffer).
ELISA of hen egg lysozyme was performed as in example 2.
The results are shown in Fig. 46. Phage derived from bacteria carrying and expressing the Fv construct fd-tet Fv
40 D1 .3 bind to the immobilised hen egg lysozyme, and when taking the phage titer into account,, indeed apparently better
than the single-chain Fv bearing phages produced by fd-tet scFv D1 .3 carrying bacteria. The specificity of the reaction
and the requirement for a functional VH domain is demonstrated by the fd-tet Fv D1.3 (AS-Stuffer) control in which
disruption of the VH domain and consequently of the Fv fragment association eliminates binding to lysozyme.
As a final control of the expected structure of the VK/genelll fusion protein a Western Blot was carried out. 20 jlxI
45 of phage suspensions concentrated 100 fold by two sequential precipitations with PEG were applied to a 10% SDS-
PAGE gel, electrophoretically separated and then transferred to a PVDF membrane (Immobilon, Millipore) in a semi-
dry Western transfer apparatus (Hoefer). Remaining binding sites on the filter were blocked by 1h incubation with 3%
BSA in PBS, and detection of the gene III protein accomplished by incubation with a 1:1000 diluted rabbit anti-genelll
antiserum for 2h, several washes in PBS/0.1% Tween 20, incubation with peroxidase-conjugated goat anti-rat immu-
ne noglobulin antibodies, washes and development with the chromogenic substrate diaminobenzidine/CoCI 2 /0.03% H 2 0 2 .
The Fv phage fd-tet Fv D1 .3 yields a band for the gene III fusion protein (data not shown), that is intermediate in size
between the bands obtained for a wild-type gene III protein from fd-DOGI and the scFv-gene III fusion protein from fd-
tet scFv D1 .3, thus proving the presence of a single immunoglobulin domain covalently fused to the gene III product
int he Fv phage.
55 in summary, Fv-gene III fusions in which one V domain is fused to the gene III protein and the other V domain
associates non-covalently can be- presented in functionally active form on the surface of filamentous phage. This
opens the possibility to genetically select for stably associated Fv fragments with defined binding specificities from V
gene libraries expressed in phages.
63
EP0 774 511 A1
Example 40 A PCR Based Technique for one step Cloning of Human V-genes as Fab Constructs
This example describes a PCR based technique to "assemble" human Fabs by splicing together the heavy and
light chain DNA with a separate piece of 'linker' DNA. A mixture of universal primers is used which should make the
5 technique applicable to all human V-genes.
The general technique for PCR assembly of human V-genes to create a Fab construct is described. The efficiency
of this technique was assessed by "assembling", cloning and expressing a human anti rhesus-D (Rh-D) Fab from a
IgG-K monoclonal hybridoma. We also demonstrate the potential to rescue human monoclonal antibodies from poly-
clonal cell populations by assembling, cloning, expressing and isolating an IgG-lambda monoclonal anti-Rh-D Fab
10 from a polyclonal lymphoblastic cell line (LCL).
The overall strategy for the PCR assembly is shown in fig. 47 and is described in more detail below. For Fab
assembly, the VH-CH1 and VK-CK or V lambda-C lambda light chains are amplified from first strand cDNA and gel
purified. Heavy and light chain DNA are then combined together with linker DNA and flanking oligonucleotides in a
new PCR reaction. This results in a full length Fab construct since the 5' end of the linker DNA is complementary to
15 the 3' end of the CH1 domain and the 3' end of the linker is complementary to the 5' end of the light chain domain. The
linker DNA contains terminal residues of the human CH1 domain, the bacterial leader sequence (pelB) for the light
chain and the initial residues of the VK or V lambda light chain (fig. 2). Finally, after gel purification, the Fab construct
is reamplified with flanking oligonucleotides containing restriction sites for cloning.
20 Oligonucleotide primers : In order to develop the PCR cloning of human V genes it was necessary to design a new
range of human specific oligonucleotide primers.
The PCR primers at the 5' end of the VH and VK and Vlambda gene exon (BACK primers) are based on sequence
data extracted from the Kabat database, (Kabat, E.A. at al, Sequences of Proteins of Immunological Interest. 4th
25 Edition. US Department of Health and Human Services. 1987) the EMBL database, the literature (Chuchana, P., at al,
Eur J. Immunol. 1990. 20:1317) and unpublished data. The sequence of the VH, VK and Vlambda primers are given
in table 1. In addition, extended VH primers with Sfil sites at the 5' end were also designed (Table 10) for adding a
restriction site after assembly.
Table 1 0 also shows the 3' primers (FORWARD primers) designed for the PCR based cloning of human V genes.
30 There are two sets of these depending on whether a Fab or scFv is to be produced. For Fab assembly, the forward
primer was based at the 3' end of the CH1 domain, CK domain and Clambda domain. In addition, the CK and C2
FORWARD primers were also synthesized as extended versions with Not1 sites at their 5' ends.
Primers complementary to the CH1 forward primers and the VkK and V lambda back primers were synthesized
to permit generation of linker DNA by PCR amplification of a plasmid template containing the Fab linker (Table 10). To
35 ensure adequate amplification, the primers were extended into the actual linker sequence.
A RNA preparation
This is essentially the same as described in Example 14, but using material of human origin. In the results given
40 in this example human hybridoma and human polyclonal lymphoblastic cell lines were used.
B cDNA preparation
Approximately 4uxj of total RNA in 20ul water was heated at 65°C for 3 minutes, quenched on ice and added to a
45 30 ul reaction mixture resulting in a 50ul reaction mixture containing 140mM KCI, 50mM Tris, HCI (pH8.1 @ 42°C),
8mM MgCI2, 10mlvl DTT, 500uM deoxythymidine triphosphate 500 uM deoxycytosine triphosphate, 500 uM deoxya-
denosine triphosphate and 500 uM deoxyguanosine triphosphate, 80 units of human placental RNAse inhibitor and
10pmol of the appropriate Forward primer (HulgG14CH1 FOR, HUlgMFOR, HuCKFOR, HuCLFOR). Two ul (50 units)
of avian myeloblastosis virus (AMV) reverse transcriptase was added, the reaction incubated at 42°C for 1 hour, heated
50 to 100°C for 3 minutes, quenched on ice and centrifuged for 5 minutes.
C Primary PCRs
For the primary PCR amplifications, an equimolar mixture of the appropriate family based BACK and FORWARD
55 primers was used. (See specific examples 40a and 40b given later in this example). A 50ul reaction mixture was
prepared containing 5ul of the supernatant from the cDNA synthesis, 20 pmol total concentration of the FORWARD
primers, 250 uM dNTPs, 50mM KCI, 100mM Tris. HCI (pH 8.3), 1.5 mM MgC12, 175ug/ml BSA and 1 ul (5 units)
Thermus aquaticus (Tag) DNA polymerase (Cetus, Emeryville, CA). The reaction mixture was overlaid with paraffin oil
64
EP0 774 511 A1
and subjected to 30 cycles of amplification using a Techne thermal cycler. The cycle was 94°C for 1 minute (denatur-
ation), 57°C for 1 minute (annealing) and 72°C for 1 minute (extension). The product was analyzed by running 5ul on
a 2% agarose gel. The remainder was extracted twice with ether, twice with phenol/chloroform, ethanol precipitated
and resuspended in 50ul of H 2 0.
5
D Preparation of linker
To make the Fab linker DNA, 13 separate PCR reactions were performed using HulgG1-4CH1 FOR and each of
the reverse VK or V lambda oligonucleotides. The template was approximately Ing of pJM-1fab D1 .3 (fig. 48) The PCR
10 reaction reagents were as described above and the cycle was 94°: 1 min, 45°:1min and 72°: 1 min. The linkers were
analyzed on a 4% agarose gel, purified on a 2% agarose gel, eluted from the gel on a Spin-X column and ethanol
precipitated.
E Assembly PCRs
15
For PCR assembly of a human Fab approximately 1 ug of a primary heavy chain amplification and 1 ug of a primary
light chain amplification were mixed with approximately 250ng of the appropriate linker DNA in a PCR reaction mixture
without primers and cycled 7 times (94°: 2 min, 72°:2.5 min) to join the fragments. The reaction mixture was then
amplified for 25 cycles (94°: 1 mi, 68°-72°:1 min, 72°:2.5 min) after the addition of 20 pmol of the appropriate flanking
20 BACK and FORWARD primers.
F Adding Restriction Sites
The assembled products were gel purified and reamplified for 25 cycles (94°: 1 min, 55°: 1 min, 72°: 25min) with
25 the flanking oligonuceotides containing the appended restriction sites. PCR buffers and NTPs were as described pre-
viously.
Specific examples of PCR assembly of human immunoglobulin genes
30 a. PCR assembly of a Fab from a human hybridoma : the human monoclonal anti Rh-D cell lines Fog-1 (IgG-k)
was derived from EBV transformation of the PBLs of a Rh-D negative blood donor immunized with Rh-D positive
blood and has been previously described (Melamed, M.D., atal., J. Immunological Methods. 1987. 104:245) (Hugh-
es-Jones N.C., at al., Biochem. J. 1990. 268:135) (Gorick, B.D. et al., Vox. Sang. 1988, 55:165) Total RNA was
prepared from approximately 1 0 7 hybridoma cells. First strand cDNA synthesis was performed as described above
35 using the primers HulgG1-4CHIFORand HUCKFOR. Primary PCRs were performed for the VH-CHI using a mixture
of the 6 HuVHBACK primers and HulgGI-4CGIFOR and for the VK-CK using a mixture of the 6 HuVKBACK primers
and HuCKFOR. A Fab construct was assembled as described above, restricted with Sfil and Notl, gel purified and
ligated into pJM-IFab D1.3 restricted with Sfil and Notl. The ligation mixture was used to transform competent E.
coli E.M.G. cells. Ninety-six clones were toothpicked into media in microtitre plate wells, grown to mid-log phase
40 at 30°C and then expression of the Fab was induced by heat shocking at 42°C for 30 min followed by growing for
4 hours at 37°C. The ninety-six clones were then screened for anti-Rh-D activity as described below.
b. assembly of human Fabs from a polyclonal (LCL) : A polyclonal LCL "OG" was derived from EBV transformation
of approximately 10 7 peripheral blood lymphocytes (PBLs) from a Rh-D negative donor immunized with Rh-D
positive red blood cells. The cells were plated at a concentration of approximately 1 0 5 cells per well. Positive wells
45 were identified by screening the cells harvested and then subcloned once. Typing of the well indicated that an IgG-
lambda antibody was being produced. At this stage, total RNA was prepared from approximately 10 6 cells. First
strand cDNA synthesis was performed as described above using the primers HulgG1-4CG1 FOR and HuCLFOR.
Primary PCRs were performed for the VH-CH1 using a mixture of the 6 HuVHBACK2 primers and HulgG1-4
CG 1 FOR and for the V lambda-C lambda using a mixture of the 7 Hu V BACK primers and HuC FOR. Restriction,
so cloning and screening proceeded as described. To determine the diversity of the clones, the VH and V lambda
genes of 1 5 clones were PCR amplified, restricted with the frequent cutting restriction enzyme. BstN1 and analyzed
on a 4% agarose gel (see example 20).
Assay for anti-Rh-D activity and demonstration of specificity : A 5% (vol/vol) suspension of either Rh-D positive (OR2R2)
55 or Rh-D negative (Orr) erythrocytes in phosphate buffered saline (PBS, pH 7.3) were incubated with a papain solution
for 10 min at 37°C. The erythrocytes were washed three times in PBS and a 1% (vol/vol) suspension of erythrocytes
was made up in PBS supplemented with 1% (vol/vol) of bovine serum albumin ( BSA). Fifty ul of a papain treated
erythrocyte suspension and 50ul of phage supernatant were placed in the wells of round bottom microtitre plates and
65
EP0 774 511 A1
the plates were placed on a Tltertek plate shaker for 2 min. After 15 min incubation at 37°C 100 ul of PBS/BSA was
added to each well. The plates were centrif uged at 200 g for 1 min and the supernatant was discarded. The erythrocytes
were resuspended in the remaining PBS/BSA and the Fab fragments were crosslinked by addition of the 9E10 mon-
oclonal antibody (50ul a 1 ug/ml solution in PBS/BSA) directed against the myc peptide tag (Ward, E.S., at al., Nature
5 1989. supra). The plates were placed at room temperature (RT) until sedimentation had occurred. Agglutination of
erthrocytes caused a diffuse button of erythrocytes and the results were evaluated macroscopically. Specificity was
confirmed with a standard prepapainized (as above) panel of 9 erythrocyte suspensions in PBS (all suspensions blood
group 0, 4 D positive and 5 D negative) known to have homozygous expression of all the clinically relevant erythrocyte
blood group alloantigens. The number of copies of the D antigen on the D positive cells varied between 10,000 and
10 20,000 per erythrocyte depending on the Rh genotype. Briefly, 50 ul phage supernatant in PBS supplemented with 2%
(vol/vol) skimmed milk was mixed with 50 ul of a 2% erythrocyte suspension in PBS in glass tubes and incubated for
15 min at 37°C. After one wash with PBS/BSA the erythrocytes were pelleted and resuspended in 50 ul donkey anti-
human lambda light chain (Sigma L9527, diluted 1 :40 in PBS/BSA). The tubes were centrifuged for 1 min at 200g and
agglutination was read macroscopically using "tip and roll" method.
15
Results
a PCR assembly of a Fab from a human hybridoma : A single band of the correct size was obtained after amplifi-
cation. Thirty-eight of 96 clones (40%) screened specifically agglutinated Rh-D positive but not Rh-D negative red
20 blood cells. The results demonstrate a high frequency of successful splicing in the assembly process and the
potential of this technique for one step cloning of human hybridomas.
b Assembly of human Fabs from a polyclonal lymphoblastic cell line (LCL) : Analysis of the diversity of the clones
indicated that 3 different heavy chain families and 2 different light chains families were present. Five anti-Rh-D
specific clones were identified out of 96 screened. The VH and VX chains had identical nucleotide sequences in
25 each clone and were typical of anti-Rh-D V-genes (unpublished results). The results demonstrate the potential of
this technique to assemble, clone and isolate human antibody fragments from polyclonal cell populations (see also
section on isolation of specific binding activities from an 'unimmunized' human library (examples 42 and 43).
Example 41
30
Selection of Phage Displaying a Human Fab Fragment directed against the Rhesus-D Antigen by binding to Cells
displaying the Rhesus D Antigen on their Surface
A large number of important antigens are integral components of cell surface membranes, i.e. they are cell surface
35 antigens. These include tumor specific antigens and red and white blood cell surface antigens. In many instances, it
would be important to isolate antibodies against these antigens. For example, antibodies directed against the rhesus-
D (Rh-D) antigen on red blood cells are used both diagnostically and therapeutically. Many of these antigens are difficult
to purify and some, like Rh-D, are not biologically active when isolated from the membrane. Thus, it would be useful
to be able to affinity purify antibody fragments displayed on the surface of bacteriophage directly on cell surface anti-
40 gens. To test the feasibility of affinity purification on cell surface antigens, the anti-Rh-D human monoclonal antibody
Fog-B was displayed as a Fab fragment on the surface of bacteriophage fd. The displayed Fog-B Fab fragment bound
antigen as determined by agglutination assay and could be affinity purified on the basis of its binding on the surface
of Rh-D positive red blood cells but not Rh-D negative red blood cells.
45 Materials and Methods
Construction of a clone encoding an anti-Rh-D Fab fragment in phagemid pHEN1 and display of the Fab fragment
on the surface of bacteriophage fd.
The human hybridoma Fog-B has been previously described (N.C. Hughes-Jones at al Biochem, J. 268 1 35 (1 990).
so it produces an IgG-l/lambda antibody which binds the Rh-D antigen. RN A was prepared from 1 0 7 hybridoma cells using
a modified method of Cathala (as described in example 14) and 1st strand cDNA synthesized using specific immu-
noglobulin heavy and light chain primers (HuVH1 FOR [example 40] and HuCX FOR (5'-GGA ATT CTT ATG AAG ATT
CTG TAG GGG CCA C-3 1 )) as described in example 14. The VH gene was subsequently amplified from an aliquot of
the 1st strand cDNA using HuVH4aBACK and HuVH1 FOR. The MX gene was amplified using a primer specific for
55 Fog-B (VMog-B, 5'-AAC CAG CCA TGG CC AGT CTG TGT TGA CGC AGO C-3'). The PCR conditions were as
described in example 40. The PCR products were analyzed by running 5uJ on a 2% agarose gel. The remainder was
extracted twice with ether, twice with phenol/chloroform, ethanol precipitated and resuspended in 50uJ of H 2 0. The
amplified VH DNA was digested with Pst1 and BstEII, and the amplified V\-C\ DNA with Ncol and EcoR1 . The frag-
66
EP0 774 511 A1
merits were purified on a 2% agarose gel, extracted using Geneclean, and sequentially ligated into the soluble expres-
sion vector pJM-1 Fab D1 .3 (Fig 48). Clones containing the correct insert were initially identified by restriction analysis
and verified by assay of expressed soluble Fab (see example 23 for induction conditions). The Fog-B Fab cassette
was amplified from pJM-1 by PCR using HUVH4BACK-Sfi and Hu C^-Not, digested with the appropriate restriction
5 enzymes and ligated into pHEN1. Clones containing the correct insert were identified initially by restriction analysis
and subsequently by assay (see example 25 for induction conditions).
Assay for soluble Fog-B Fab fragment and phage displayed Fog-B Fab fragment for anti-Rh-D activity and docu-
mentation of specificity.
Assay of the soluble expressed Fab was performed on unconcentrated E.coli supernatant. Assay of Fog-B dis-
10 played on the phage surface was performed on phage that had been concentrated 10 fold by PEG precipitation and
then resuspended in PBS. the assays for activity and specificity are as described in example.
Cell surface affinity purification of phage displaying Fog-B anti-Rh-D Fab fragment
Purified Fog-B phage was mixed with purified phage Fd-Tet CAT-1 displaying the anti-lysozyme scFv D1.3
(pAbD1 .3) in a ratio of approximately 1 Fog-B:50 scFcDI .3. Prepapainized erythrocytes (OR2R2 [Rhesus positive] or
15 Orr [Rhesus negative]) were suspended in PBS supplemented with 2% skimmed milk powder in a concentration of
4x107/ml. One ml of this suspension was mixed with 10 11 phage suspended in 2 ml of PBS supplemented with 2%
skimmed milk and incubated for 30 min at room temperature under continuous rotation. The erythrocytes were washed
three times with an excess of ice-cold PBS (10 ml per wash) and subsequently pelleted. The phage were eluted from
the cells by resuspending in 200 uJ of 76 mM citric acid pH 2.8 in PBS for 1 min. The cells were then pelleted by
20 centrifugation for 1 min at 3000 rpm and the supernatant containing the eluted phage was neutralized by adding 200
jlxI of 240 mM Tris-base, 22mM Disodium hydrogen phosphate in 1% w/vol albumin. Serial dilutions of the eluate was
used to infect TG1 cells. Fog-B Fab phage were selected on ampicillin plates and scFcD1.3 phage on tetracycline
plates and the titre of each determined prior to selection, after selection on rhesus-D negative cells and after selection
on rhesus-D positive cells.
25
Results
Fog-B Fab fragment displayed on the surface of the phage derived from the phagemid pHEN clone specifically
agglutinated rhesus-D positive but not rhesus D-negative red blood cells. Affinity purification of the Fog-1 Fab phagemid
30 on Rh-D positive red blood cells resulted in an enrichment from 1 :50 to 1500:1 (Fog-B Fab:scFvD1 .3), whereas puri-
fication on Rh-D negative red blood cells demonstrated essentially no enrichment (10 fold).
TITRE
RATIO
Fog-B Fab scFvD1.3
Fog-B FAb/scFvD1.3
Prior to selection
1. Ox 10 8 5.0x 10 9
1:50
Selection on Rh-D negative cells
2.0 x 10 4 1.0 x 10 5
1:5
Selection on Rh-D positive cells
6.0 x 10 6 4.0 x 10 3
1500:1
Example 42 A PCR Based Technique for One Step Cloning of Human scFv Constructs
Assembly of human scFv is similar to the assembly of mouse scFvs described in example 1 4. To develop the PCR
cloning of human V genes it was necessary to design a new range of human specific oligonucleotide primers (table
10). The use of these primers for the generation of human Fabs is described in example 40. The assembly of human
scFvs is essentially the same but requires a set of FORWARD primers complementary to the J segments of the VH,
VKand V lambda genes. (For Fabs FORWARD primers complementary to the constant region are used.) The J segment
specific primers were designed based on the published JH, JK and J lambda sequences (Kabat, E.A. at al, Sequences
of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987).
In addition, a different linker is needed for scFvs than for Fabs so for human scFvs a new set of primers was
needed to prepare the linker. Primers complementary to the JH forward primers and the VK and V lambda back primers
were synthesized to permit generation of linker DNA by PCR amplification of a plasmid template containing the scFv
linker (Table 1 0, Fig. 49). To ensure adequate amplification, the primers were extended into the actual linker sequence.
Using these primers to make the scFv linker DNA, 52 separate PCR reactions were performed using each of the 4
reverse JH primers in combination with each of the 13 reverse VK and V lambda oligonucleotides. The template was
approximately Ing of pSW2scD1.3 (Ward, E.S. 1989 supra) containing the short peptide (Gly4Ser)3 ( Huston, J.S. at
al., Gene 1989. 77:61)
67
EP0 774 511 A1
A specific example of PCR assembly of a human scFv library
This example describes the generation of a human library of scFvs made from an unimmunized human:
500ml of blood, containing approximately 10 8 B-cells, was obtained from a healthy volunteer blood donor. The
5 white cells were separated on Ficoll and RNA was prepared as described in example 14.
Twenty percent of the RNA, containing the genetic material from approximately 2 x 1 0 7 B-cells, was used for cDNA
preparation as described in example 40. Heavy chains originating from IgG and IgM antibodies were kept separate by
priming cDNA synthesis with either an IgG specific primer (HulgGI -4CH1 FOR) or an IgM specific primer (HulgMFOR).
Aliquots of the cDNA was used to generate four separate scFv libraries (IgG-K, IgG-lambda, IgM-K and IgM-lambda)
10 as described in example 40. The resulting libraries were purified on 1 .5% agarose, electroeluted and ethanol precipi-
tated. For subsequent cloning, the K and lambda libraries were combined giving separate IgG and IgM libraries.
Cloning of the library : The purified scFv fragments (1-4ug) were digested with the restriction enzymes Notl and either
Sfil or Ncol. After digestion, the fragments were extracted with phenol/chloroform, ethanol precipitated. The digested
fragments were ligated into either Sfil-Notl or Ncol-Notl digested, agarose gel electrophoresis purified pHEN1 DNA
15 (6ug) (see example 24), in a 100 jlxI ligation mix with 2,000 U T4 DNA ligase (New England Biolabs) overnight at room
temperature. The ligation mix was purified by phenol extraction and ethanol precipitated. The ligated DNA was resus-
pended in 1 0 jlxI of water, and 2.5 jlxI samples were electroporated into E.coli TG1 (50 jlxI). Cells were grown in 1 ml SOC
for 1 hr and then plated on 2 x TY medium with 100 |ag/ml ampicillin and 1% glucose (AMP-GLU), in 243 x 243 mm
dishes (Nunc). After overnight growth colonies were scraped off the plates into 1 0 ml 2 x TY containing AMP-GLU and
20 15% glycerol for storage at -70°C as a library stock.
Cloning into Sfil-Notl and Ncol-Notl digested pHEN1 yielded libraries of 10 7 and 2 x 10 7 clones respectively for
the IgM libraries and approximately 5 x 10 7 clones for each of the two IgG libraries.
Example 43 Isolation of binding activities from a library of scFvs from an unimmunized human
25
The ability to select binding activities from human antibody libraries displayed on the surface of phage should
prove even more important than isolation of binding activities from murine libraries. This is because the standard way
of generating antibodies via hybridoma technology has not had the success with human antibodies that has been
achieved with mouse. While in some instances it will be possible to make libraries from immunized humans, in many
30 cases, it will not prove possible to immunize due to toxicity or lack of availability of an appropriate immunogen or ethical
considerations. Alternatively, binding activities could be isolated from libraries made from individuals with diseases in
which therapeutic antibodies are generated by the immune response. However, in many cases, the antibody producing
cells will be located in the spleen and not available in the circulating pool of peripheral blood lymphocytes (the most
easily accessible material for generating the library). In addition, in diseases associated with immunosuppression,
35 therapeutic antibodies may not be produced.
An alternative approach would be to isolate binding activities from a library made from an unimmunized individual.
This approach is based on estimates that a primary repertoire of 10 7 different antibodies is likely to recognize over
99% of epitopes with an affinity constant of 10 5 M" 1 or better. (Pewrelson, A.S. Immunol. Rev. (1989) 110:5). While this
may not produce high affinity antibodies, affinity could be boosted by mutation of the V-genes and/or by using the
40 isolated VH domain in a hierarchical approach with a library of light chains (or vice verse). In this section" we demon-
strate the feasibility of this approach by isolating specific antigen binding activities against three different antigens from
a library of scFvs from an unimmunized human.
Materials and Methods
45
The generation of the human scFv library used for the isolation of binding activities described in this example is
detailed in example 42.
Estimation of diversity of original and selected libraries : Recombinant clones were screened before and after selection
by PCR (example 20) with primers LMB3 (which sits 5' of the pelB leader sequence and is identical to the reverse
50 sequencing primer (-40 n) of pUC1 9) and fd-SEQ1 (see example 37) followed by digestion with the frequent-cutting
enzyme BstNI. Analysis of 48 clones from each unselected library indicated that 90% of the clones had inset, and the
libraries appeared to be extremely diverse as judged by the BstNI restriction pattern.
Rescue of Phagemid libraries for enrichment experiments : To rescue phagemid particles from the library, 100 ml 2 x
TY containing AMP-GLU (see example 42) was inoculated with 1 0 9 bacteria taken from the library (prepared in example
55 42) (approx. 10 uJ) and grown for 1.5 hr, shaking at 37°C. Cells were spun down (IEC- centrifuge, 4 K, 15 min) and
resuspended in 1 00 ml prewarmed (37°C) 2x TY-AMP (see example 41 ) medium, 2 x 1 0 10 pfu of VCS-M1 3 (Stratagene)
particles added and incubated 30 min at 37° without shaking. Cells were then transferred to 900 ml 2 x TY containing
ampicillin (100 |ag/ml) and kanamycin (25 |ag/ml) (AMP-KAN), and grown overnight, while shaking at 37°C. Phaae
68
EP0 774 511 A1
particles were purified and concentrated by three PEG-precipitations (see materials and methods) and resuspended
in PBS to 10 13 TU/ml (ampicillin resistant clones).
Enrichment for phOx:BSA binders by selection on tubes : For enrichment, a 75 x 1 2 mm Nunc-immunotube (Maxisorp;
Cat. No. 4-44202) was coated with 4 ml phOx:BSA (1 mg/ml; 14 phox per BSA in 50 mM NaHC03 pH 9.6 buffer)
5 overnight at room temperature. After washing three times with PBS, the tube was incubated for 2 hr at 37°C with PBS
containing 2% Marvel (2% MPBS) for blocking. Following three PBS washes, phagemid particles (10 13 TU) in 4 ml of
2% MPBS were added, incubated 30 min at room temperature on a rotating turntable and left for a further 1 .5 hours.
Tubes were then washed with 20 washes of PBS, 0.1% Tween 20 and 20 washes PBS (each washing step was
performed by pouring buffer in and out immediately). Bound phage particles were eluted from the tube by adding 1 ml
10 1 00 mM triethylamine pH 11.5 and rotating for 1 5 min. The eluted material was immediately neutralised by adding 0.5
ml 1 .0 M Tris-HCI, pH 7.4 and vortexed. Phage was stored at 4°C.
Eluted phage (in 1 .5 ml) was used to infect 8 ml logarithmic growing E.coli TG1 cells in 15-ml 2 x TY medium, and
plated on AMP-GLU plates as above yielding on average 10 7 phage infected colonies.
For selection of phOx:BSA binders, the rescue-tube enrichment -plating cycle was repeated 4 times, after which
15 phagemid clones were analysed for binding by ELISA. Enrichment for lysozyme binders by panning and on columns :
A petri dish (35 x 10 mm Falcon 3001 Tissue culture dish) was used for enrichment by panning. During all steps, the
plates were rocked on an A600 rocking plate (Raven Scientific). Plates were coated overnight with 1 ml turkey egg
white lysozyme (3 mg/ml) in 50 mM sodium hydrogen carbonate (pH 9.6), washed three times with 2 ml PBS, and
blocked with 2 ml 2% MPBS at room temperature for 2 hours. After three PBS washes approximately 10 12 TU phage
20 particles in 1 ml 2% MPBS were added per plate, and left rocking for 2 hr at room temperature. Plates were washed
for 5 min with 2 ml of the following solutions: 5 times PBS, PBS-Tween (0.02% Tween-20), 50 mM Tris-HCI (pH 7.5)
+ 500 mM NaCI, 50 mM Tris-HCI (pH 8.5) + 500 mM NaCI, 500 mM Tris-HCI (pH 9.5) + 500 mM NaCI and finally 50
mM sodium hydrogen carbonate pH 9.6 Bound phage particles were then eluted by adding 1 ml 100 mM triethylamine
pH 11.5 and rocking for 5 min before neutralising with 1 M tris-HCI (pH 7.4) (as above). Alternatively, 1 ml turkey egg
25 white lysozyme-Sepharose columns were used for affinity purification (McCafferty, J., It al., Nature 1990. 348: 552)
Cclumns were washed extensively with PBS, blocked with 15 ml 2% MPBs, and phage (10 12 TU) in 1 ml 2% MPBS
loaded. After washing with 50 ml PBS, 10 ml PBS- Tween (PBS + 0.02% Tween-20), 5 ml of 50 mM Tris-HCI (pH 7.5)
- 500 mM NaCI, 5 mM Tris-HCI 9pH 8.5) + 500 mM NaCI, 5ml of 50 mM Tris-HCI (pH 9.5) + 500 mM NaCI and finally
5 ml of 50 mM sodium hydrogen carbonate pH 9.6. Bound phage was eluted using 1.5 ml 100 mM triethylamine and
30 neutralised with 1 M Tris-HCI (pH 7.4).
For selection of turkey egg white lysozyme binders, the rescue-tube enrichment-plating cycle or rescue-column-
plating cycle was repeated 4 times, after which phagemid clones were -analysed for binding by ELISA.
Rescue of individual phagemid clones for ELISA : Clones resulting from reinfected and plated phage particles eluted
after 4 rounds of enrichment, were inoculated into 150 uJ of 2 x TY-AMP-GLU in 96-well plates (cell wells, Nunclon),
35 grown with shaking (250rpm) overnight at 37°C. A 96-well plate replicator ('plunger') was used to inoculate approxi-
mately 4 jlxI of the overnight cultures on the master plate into 200 uJ fresh 2 x TY-AMP-GLU. After 1 hr, 50 jlxI 2 x TY-
AMP-GLU containing 1 0 8 pfu of VCS-M1 3 was added to each well, and the plate incubated at 37°C for 45 min, followed
by shaking the plate at 37°C for 1 hr. Glucose was then removed by spinning down the cells (4K, 1 5 min), and aspirating
the supernatant with a drawn out glass pasteur pipet. Cells were resuspended in 200 uJ 2 -x TY-AMP-KAN (Kanamycin
40 50 ug/ml) and grown 20 hr, shaking 37°C. Unconcentrated supernatant containing phage was taken for analysis by
ELISA.
ELISA
45 Analysis for binding to phOx:BSA, BSA or lysozyme was performed by ELISA (see example 9), with 100 u.g/ml
phOx: BSA or BSA, or 3 mg/ml turkey egg white lysozyme used for coating. Determination of cross reactivity to unrelated
antigens with the isolated clones was also determined by ELISA on plates coated with 1 00 ug/ml of an irrelevant antigen
(keyhole limpet haemocyanin (KLH), ovalbumin, chymotrypsinogen, cytochrome C, thyroglobulin, GAP-DH (glyceral-
dehyde-3-phosphate dehydrogenase), or trypsin inhibitor).
50 Characterization of ELISA positive clones : All antigen specific clones isolated were checked for cross reactivity against
a panel of irrelevant antigens as described above. The diversity of the clones was determined by PCR screening as
described above and at least two clones from each restriction pattern were sequenced by the dideoxy chain termination
method.
55 Results
Isolation and characterization of phQx:BSA binders : After 4 rounds of selection, ELISA-positive clones were iso-
lated for phOx:BSA. All clones originated from the IgM library. Of 96 clones analysed, 43 clones were binding to both
69
EP0 774 511 A1
phOx:BSA and BSA, with ODs ranging from 0.4 to 1.3 (background 0.125). These clones are designated as BSA
binders. The binding to BSA seemed to be specific, since none of the 11 clones analysed gave a signal above back-
ground when used in an ELISA with KLH, ovalbumin, chymotrypsinogen, cytochrome C. lysozyme, thyroglobulin, GAP-
DH, or trypsin inhibitor, all BSA binding clones had the same BstNl restriction pattern, and 14 clones were completely
5 sequenced. Thirteen of the fourteen clones had the same sequence, the VH was derived from a human VH3 family
gene and the VL from a human V lambda 3 family gene (Table 1). The other BSA binder was derived from a human
VH4 family gene and a human Vkl family gene (data not shown).
One clone was isolated which bound to phox:BSA only (OD 0.3), and bound phage could be completed off com-
pletely by adding 0.02 mM 4-E-amino-caproic acid methylene 2-phenyl-oxazol-5-one (phOx-CAP) as a competitor.
10 Also no binding above background could be detected to the panel of irrelevant proteins described above. The sequence
revealed a VH derived from a human VH 1 family gene and a VL derived from a human V lambda 1 family gene (Table 11).
Isolation and characterisation of lysozyme binders : After 4 rounds of selection, 50 ELISA-positive clones were isolated
for turkey lysozyme. The majority of the clones, greater than 95%, were from the IgM library. The binding to lysozyme
seemed to be specific, since none of the clones analysed gave a signal above background when used in an ELISA
15 with KLH, ovalbumin, chymotrypsinogen, cytochrome C, thyroglobulin, GAP-DH, or trypsin inhibitor. The lysozyme
binding clones gave 3 different BstNl restriction patterns, and. at least 2 clones from each restriction pattern were
completely sequenced. The sequences indicated the presence of 4 unique human VH-VL combinations. (Table 11).
Conclusion
20
The results indicate that antigen binding activities can be isolated from repertoires of scFvs prepared from IgM
cDNAfrom human volunteers that have not been specifically immunized.
Example 44
25
Rescue of human IgM library using helper phage lacking gene 3 (5g3)
This example describes the rescue of gene 3 fusions from a human library using a helper phage with a gene 3
deletion.
30 100uJof bacterial stock of the IgM phagemid library prepared as described (example 42), containing 5x1 0 8 bacteria,
was used to inoculate 100mls of 2xTY medium containing 100|ug/ml ampicillin, 2% glucose (TY/Amp/Glu). This was
grown at 37°C for 2.5 hours. 1 0 mis of this culture was added to 90 mis of prewarmed TY/Amp/Glu and infection carried
out by adding 10mls of a 200 fold concentrate of K07 helper phage lacking gene 3 (M1 3K07glll A No.3) (example 34)
and incubating for 1 hour at 27°C without shaking. Preparation of M13K07glll No.3 was as described in example 34.
35 After centrifugation at 4,000 r.p.m. for 10 minutes the bacteria were resuspended in 1 00 mis of 2 xTY medium containing
100 uxj/ml ampicillin (with no glucose). Titration of the culture at this point revealed that there were 1.9x10 8 infected
bacteria as judged by their ability to grow on plates containing both ampicillin (100jag/ml) and kanamycin (50ujg/ml).
Incubation was continued for 1 hour with shaking before transferring to 2.5 litres of 2xTY medium containing 100|ag/
ml ampicillin, 50|ug/ml kanamycin, contained in five 2.5 litre flasks. This culture was incubated for 16 hours and the
40 supernatant prepared by centrifugation. (10-15 minutes at 10,000 r.p.m. in a Sorvall RCSB centrifuge at 4°C). Phage
particles were harvested by adding 1/5th volume of 20% polyethylene glycol, 2.5 M NaCI, standing at 4°Cfor 30 minutes
and centrifuging as above. The resulting pellet was resuspended in 40mls of 10mM Tris, 0.1mm EDTA pH 7.4 and
bacterial debris removed by centrifugation as above. The packaged phagemid preparation was then re-precipitated,
collected as above and resuspended in 10mls of 10mM Tris, 0.1 mM EDTA pH 7.4. The litre of this preparation was
45 4. 1x1 0 13 transducing units/ml (ampicillin resistance).
Tubes coated with OX-BSA were prepared as described in example 45 for panning the phagemid library from
example 42. The rescued library was also panned against tubes coated with bovine thyroglobulin (Sigma). These were
coated at a concentration of 1mg/ml thyroglobulin in 50mM NaHC03 pH9.6 at 37°C, overnight. Tubes were blocked
with PBS containing 2% milk powder (PBS/M) and incubated with 1 ml of the rescued phagemid library (the equivalent
50 of 250mls of culture supernatant) mixed with 3mls of PBS/M for 3 hours. Washing, elution, neutralisation and infection
were as described in example 45.
Results: Panning against oxazalone - BSA
55 The first round of panning against OX-BSA yielded 2.8x1 0 6 phage. A large bacterial plate with 1 .4x1 0 6 colonies
derived from this eluate was scraped into 10mls of 2xxTY, 20% glycerol, shaken for 10 minutes, aliquoted and stored.
This was also used to inoculate a fresh culture for rescue with M1 3K07glll No.3. (Bacteria and rescued phage derived
from first round panning against OX-BSA are named OXPAN1. Bacteria or rescued phage derived from second and
70
EP0 774 511 A1
third round panninas are named OXPAN2 and OXPAN3 respectively) Rescue of phagemid with M1 3K07glll No. 3 after
each round of panning was essentially as described above but using 5ml volumes for the initial cultures in TY/Amp/
Glu, using 1ml of helper phage and transferring to 100-500mls of 2xTY medium containing 100jag/m I ampicillin, SOjixg/
ml kanamycin. Second and third round panning steps were as described above for the first round, but using 0.8-1 .Omls
5 of 1 00 fold concentrated phage (the equivalent of 80-1 00 mis of culture supernatant). The eluate from the second round
panning contained 8x1 0 8 infectious particles and the eluate from the third round panning contained 3.3x1 0 9 infectious
particles.
Panning against thyroglobulin
10
The first round panning against thyroglobulin yielded 2.52x1 0 5 infectious particles. Half of the eluate was used to
generate 1 .26x1 0 5 bacterial colonies on a large plate. These colonies were scraped into 10mls of 2xTY, 20% glycerol,
shaken for 10 minutes, aliquoted and stored. These bacteria and rescued phage derived from them are termed
THYPAN1 , and used to inoculate a fresh culture for rescue with M1 3K07gl 1 1 No.3 to give a polyclonal rescued phage
15 preparation. Material similarly derived from second and third round pannings are termed THYPAN2 and THYPAN3
respectively. Second and their round pannings with thyroglobulin were as described for second and third round OX-
BSA panning. The eluate from the second round panning contained 8x1 0 7 transducing units and the eluate from the
third round panning contained 6x1 0 7 infectious particles.
20 ELISA screening of clones derived by panning
40 colonies derived form the third round of panning against thyroglobulin (THYPAN3) were picked into a 96 well
plate and grown overnight at 37°C in 200uJ of TY/Amp/Glu. Similarly 48 colonies from two rounds and 48 colonies from
three rounds of panning against OX-BSA were grown (OX-PAN2 and OX-PAN3). Polyclonal phage were prepared at
25 the same time. Next day 5uJ from each culture was transferred to 1 OOuJ of fresh prewarmed TY/Amp/Glu grown for 1 .5
hours and M13K07glll No.3 added (2 x 10 5 infectious phage per well in 1 OOjllI of TY/Amp/Glu). these were incubated
for 1 hour at 37°C without shaking, centrifuged at 4,000 r.p.m. for 10 minutes, resuspended in 1 SOjllI of 2xTY medium
containing 100|ag/ml ampicillin and incubated for afurther hour with shaking before adding to 2mls of medium containing
100|ug/ml ampicillin, 50uxj/ml kanamycin. After overnight growth the cultures were centrifuged at 4,000 r.p.m. for 10
30 minutes and the supernatants collected. ELISA plates used to screen THYPAN3 clones were coated at 37°C overnight
with 200jag/ml thyroglobulin in 50mM NaHC03pH9.6. Plates used for OXPAN2 and OXPAN3 were coated at lOOjug/
ml OX-BSA in PBS at 37°C overnight.
1 20uJ of culture supernatant was mixed with 30uJ of 5x PBS, 1 0% milk powder and incubated at room temperature
for 2 hours at room temperature. ELISAs were carried out as described in example 18.
35 For thyroglobulin, 1 8 out of 40 clones were positive (0.3-2.0 O.D. after 30 minutes). (A phage control (vector pCAT3)
gave a reading of 0.07 O.D.). In addition, positives were also seen on the polyclonal phage preparations THYPAN1
(0.314 O.D.) and THYPAN2 (0.189 O.D.) compared with phage derived from the original non-panned phagemid library
(0.069 O.D.). All polyclonal phage were PEG precipitated and used at a 10 fold concentration.
PCR reactions and BstN1 digests were carried out on the positive clones as described above and six different
40 patterns of DNA fragments were obtained showing that at least six different clones had been isolated.
For OX-BSA after two rounds of panning, 30 of 48 clones were positive by ELISA and after three rounds , 42 of
48 were positive. In a separate experiment, positive signal was obtained from the polyclonal phage preparations
OXPAN1 (0.988 OD) and OXPAN2 (1 .717 OD) compared with phage derived from the original non-panned phagemid
library (0.186 O.D.) after 30 minutes.
45
Specificity of clones for thyroglobulin or OX-BSA
Selected clones (11 anti-thyroglobulin, 5 anti-OX-BSA) representing each of the different BstNl restriction digest
patterns were assayed for binding to a panel of irrelevant antigens. ELISA plates were coated with antigen (100 jLxl/ml
50 in 50 mM NaHC03, pH 9.6) by overnight-incubation at 37°C. The panel of antigens consisted of keyhole limpet haemo-
cyanin, hen egg lysozyme, bovine serum albumin, ovalbumin, cytochrome c, chymotrysinogen, trypsin inhibitor, GAP-
D11 (glyceraldehyde-3-phosphate dehydrogenase), bovine thyroglobulin and oxazolone-BSA. Duplicate samples of
phage supernatant (80 uJ + 20 uJ 5 x PBS, 10% milk powder) were added to each antigen and incubated for 1 hour at
room temperature, the ELISA was carried out as described in example 18.
55 Each of the thyroglobulin specific clones (1 1 from 1 1 ) were positive for thyroglobulin (OD 0. 1 2 - 0.76) but after 60
minutes showed no binding (OD<0.03) to any of the 9 irrelevant antigens. Similarly of the 5 OX-BSA specific clones 3
had an OD 0.07 - 0.52 compared to ODs < 0.02 for the irrelevant antigens. None of the 5 clones had any binding to
BSA alone.
71
EP0 774 511 A1
Thus positive clones can be isolated after only two rounds of panning by rescuing with M1 3K07glll No.3. In addition
there is a greater likelihood with this helper of generating phage particles with more than one intact antibody molecule.
This will potentially increase the avidity of phage-antibodies and may enable isolation of clones of weaker affinity.
s Example 45: Alteration of fine specificity of scFv D1 .3 displayed on phage by mutagenesis and selection on immobilised
turkey lysozyme
The D1 .3 antibody binds hen egg lysozyme (HEL) with an affinity constant of 4.5 x 10 7 M" 1 whereas it binds turkey
egg lysozyme (TEL) with an affinity of <1 x1 0 5 M" 1 . (Harper et al (1 987) Molecular Immunology 24 p97-1 08, Amit et al
10 (1 986) Science 233 p747-753).
It has been suggested that this is because the glutamine residue present at position 121 of HEL (gln1 21 ) is rep-
resentated by histidine residue at the same position in TEL. Thus mutagenising the D1.3 antibody residues which
interact with gln1 21 of HEL may facilitate binding to TEL.
According to Amit at al, supra, tyrosine at amino acid position 32, phenylalanine at position 91 and tryptophan at
15 position 92 of the light chain interact with gln1 21 of HEL. In addition tyrosine at position 101 of the heavy chain also
Interacts. None of these residues are predicted to be involved in determining the main chain conformation of the an-
tibody variable regions (Chothia and Lesk (1987) Journal of Molecular Biology 196, p901-917).
Mutagenesis of pCAT3SCFvD1 .3
20
The oligonucleotides mutL91,92, was prepared too randomise phenylalanine at position 91 (L91) and tryptophan
at position 92 (L92) of the light chain. The oligonucleotides mutL32, was prepared to randomise tyrosine at light chain
position 32 (L32) and the oligonucleotides mutH101 was prepared to randomise tyrosine at position 101 of the heavy
chain (H101). mutL91,92:
25
5' CGT CCG AGG AGT ACT NNN NNN ATG TTG ACA GTA ATA 2'
mutL32 :
5 ' CTG ATA CCA TGC TAA NNN ATT GTG ATT ATT CCC 3 '
30 mutHlOl:
3 1 CCA GTA GTC AAG CCT NNN ATC TCT CTC TCT GGC 3 *
(N represents a random insertion of equal amounts of A,C,G or T) in vitro mutagenesis of the phagemid vector,
pCAT3scFcD1 .3 ( example 17) with the oligonucleotide mutL91,92 was carried out using an in vitro mutagenesis kit
35 (Amersham). The resultant DNA was transformed by electroporation into TG1 cells using a Bio-Rad electroportor.
78,000 clones were obtained and these were scraped into 15mls of 2xTY/20% glycerol. This pool was called
D1 .3L91 L92. Single stranded DNA was prepared by rescue with M1 3K07 as described in Sambrook et al, 1 989 supra,
and sequenced with the primer FDTSEQ1 , using a Sequenase sequencing kit (United States Biochemical Corporation).
This revealed that the DNA had been successfully mutagenised as judged by the presence of bands in all four
40 DNA sequencing tracks at the nucleotide positions encoding L91 and L92. This mutagenised single stranded DNA was
subjected to a further round of mutagenesis as above using either mutL32 or mutH101 oligonucleotides. Mutagenesis
with mutL32 gave rise to 71,000 clones (pool called D1.3L32) while mutH101 gave 102,000 clones (pool called
D1.3H101). These clones were scraped into 15mls of 2xTY/20% glycerol. Single stranded DNA derived from each
pool was sequenced with the oligonucleotides D1 .3L40 and LINKSEQ1 respectively, as described above, and shown
45 to be correctly randomised.
D1.3L40:
5 1 CAG GAG CTG AGG AGA TTT TCC 3 '
50 LINKSEQ1 :
5 * TCC GCC TGA ACC GCC TCC ACC 3 1
Preparation of rescued phage for affinity purification
55 10-20uJ of bacteria derived from each mutagenised pool (plate scrapes) was used to inoculate 5mls of TY/Glu/
Amp. All bacterial growth was at 37°C. After 2-3 hours growth, 1 ml was diluted in 5mls of prewarmed TY/Glu/Amp and
infected by addition of 0.5 mis of a 200 fold concentrate of the M13K07glll A No.3 preparation described in example
34. After 1 hour of infection the cultures were centrifuged at 4,000 r.p.m. for 10 minutes, resuspended in 2xTY, 100u.g/
72
EP0 774 511 A1
ml ampicillin, incubated for a further hour, transferred to 500 mis of 2xTY medium containing 100 |LLg/ml ampicillin, 50
|ug/ml kanamycin and grown for 16 hours. The remaining steps of phage preparation were as described in example
44. Phage were finally dissolved in lOmMTris, 1 mM EDTA pH7.4 at 1/1 00th the original culture volume.
s Affinity purification
10mls of turkey egg lysozyme at a concentration of 10mg/ml in 0.1 M NaHC03, O.SMNaCI pH8.3 was mixed with
an equal volume of swollen Cyanogen Bromide Activated Sepharose 4B (Pharmacia), covalently linked and washed
according to manufacturers instructions. Before use this matrix (TEL-Sepharose) was washed with 100 volumes of
10 PBS followed by 10 volumes of PBSM. The TEL-Sepharose was resuspended in an equal volume of PBSM and 1ml
was added to 1ml of a 50 fold concentrate of phage in PBSM and incubated on a rotating platform for 30 minutes at
room temperature. The actual phage used for this step was prepared by mixing equal volumes of the independent
preparations of the three randomised pools (D1 .3L91 92, D1 .3H1 01 and D1 .3L32). After this binding step, the suspen-
sions were loaded onto a disposable polypropylene column (Poly-Prep columns, Bio-Rad) and washed with 200 vol-
15 umes of PBS containing 0.1% Tween 20. Bound phage were eluted with 1ml of 100mM triethylamine and neutralised
with 0.5ml 1 M Tris (pH7.4). A dilution series was prepared from the eluate and used to infect TG1 cells and plated out
on TY plates containing 100|ag/ml ampicillin, 2% glucose. Plates carrying approximately 10° colonies were scraped
into 3mls of 2xTY, 20% glycerol and stored at -70°C. 1 OjlxI of this was used to initiate a second round culture which was
rescued with M13K07glllA No. 3 as described above (using a final culture volume of 100mls). Second and third round
20 affinity column purification steps were carried out as described above for the first round.
Analysis by ELISA
40 colonies derived from the third round of column purification on TEL-Sepharose were picked into a 96 well plate
25 and grown overnight at 37°C in 200uJ of TY/Amp/Glu. Phagemid particles were rescued and prepared for ELISA as
described in example 18. ELISA plates were coated overnight at 37°C with hen egg lysozyme (HEL) or turkey egg
lysozyme (TEL) at a concentration of 200uxj/ml in 50mM NaHC0 3 pH9.6 ELISAs were carried out as described in
example 18.
After 1 5 minutes incubation in substrate, 1 3 clones were found to be negative (OD<0.05 on HEL and TEL). In all
30 positives, a signal of 0. 1 -0.78 was scored on HEL with the exception of one where signal on HEL was 0.078 but signal
on TEL (OD 0.169) brought it in to the positive group. The control phagemid preparation had a percentage ratio of
signal TEL: HEL of 22%. Clones were deemed to have an unaltered binding if the ratio of TEL:HEL was less than 40%.
9 clones fell into this category. 1 8 samples were scored as having altered binding with a ratio of signal on TEL:HEL of
between 40-200%.
35 A dilution series was made on 1 0 clones which were analysed by ELISA in 6 of these clones the profile of binding
to HEL was the same as the original clone (pCAT3SCFvD1 .3) while the signal with TEL was increased (see figure 50
clone B1 ). In the remaining 4 clones, the increased signal with TEL was accompanied by a decrease in signal on HEL
(see figure 50 clone A4).
40 Competition with soluble antigen
All of the isolated clones retained binding to HEL to varying extents. In order to determine whether a soluble antigen
could compete with the immobilised antigen, a parallel experiment was carried out, as above, but with the addition of
hen egg lysozyme (1mg/ml) to TEL-Sepharose before incubating with the phage preparation. This experiment was
45 carried through 3 rounds of column purification and 40 colonies were picked. None of these clones bound HEL or GEL
demonstrating that the soluble antigen had been successful in competing out binding to the immobilised antigen.
Example 46
50 Modification of the Specificity of an Antibody by Replacement of the VLK Domain by a VLK Library derived from an
Unimmunised Mouse
When an antibody specificity is isolated it will often be desirable to alter some of its properties particularly its affinity
or specificity. This example demonstrates that the specificity of an antibody can be altered by use of a different VL
55 domain derived form a repertoire of such domains. This method using display on phage would be applicable to im-
provement of existing monoclonal antibodies as well as antibody specificities derived using phage antibodies. This
example shows that replacement of the VL domain of scFcDI .3 specific for Hen eggwhite lysozyme (HEL) with a library
of VL domains allows selection of scFv fragments with bind also to Turkey eggwhite lysozyme (TEL). More generally
73
EP0 774 511 A1
this experimental approach shows that specificities of antibodies can be modified by replacement of a variable domain
and gives a further example of the hierarchical approach to isolating antibody specificities.
The D1.3 heavy chain was amplified from an existing construct (pSW1-VHD1 .3, Ward et al., 1989 supra) by PCR
using the primers VH1 BACK and VH1 FOR, the light chain library was amplified from a cDNA library derived from the
5 spleen of an unimmunised mouse, which was synthesized by using the MJKFONX primers 1 ,2,4,5 for the first strand
as in example 14. The subsequent amplification was performed with the same forward primers and the VK2BACK
primer. The PCR assembly of the D1 .3 heavy chain with the light chain library was mediated by the signal chain Fv
linker as described in example 14.
Cloning the assembled PCR products (scFv sequences) was done after an additional PCR step (pull-through)
10 using a BACK primer providing an ApaLI site and forward primers which contained a Not 1 site as described in example
14. Apal_1/Not 1 digested PCR fragments were cloned into the similarly digested vector fdCAT2 as in example 11.
5x1 0 5 transformations were obtained after electroporation of the ligation reaction into MC1061 cells.
Screening of the phage library for TEL binders was performed by panning. Polystyrene Falcon 2058 tubes were
coated (1 6 hrs) with 2 ml of TEL-PBS (3 mg/ml) and blocked for 2 hrs with 4 ml MPBS (PBS containing 2% skimmed
15 milk powder). Phage derived from the library (5x1 0 10 transducing unites) in 2 ml of MPBS (2%) were incubated in these
tubes for 2 hrs at room temperature. The tubes were washed 3x with PBS, 1x with 50 mM Tris-HCI, pH 7.5, 0.5 M
NaCI; 1x with 50mM Tris-HCI, pH8.5, 0.5 M NaCI, 50 mM Tris-HCI, pH 9.5 M NaCI. Finally phage were eluted with 100
mM triethylamine. Eluted phages were taken to infect TG1 cells, the cells were plated on 2xTY plates containing 15
|ug/ml tetracycline and grown for 16h. The colonies were scraped into 25ml of 2xTy medium and the phages were
20 recovered by PEG precipitation. After a second round of selection for TEL binders ELISAs were performed as described
(example 2).
Analysis of 100 clones from the library before affinity selection by ELISA on plates coated with TEL showed no
binders. In contrast, after two rounds of selection for TEL binding phages about 10% of the phage clones showed
positive ELISA signals. ELISA signals were scored positive with values at least two fold higher than the fdCAT2 vector
25 without insert. A more detailed analysis of binding properties of TEL binding phages is shown in figure 51 .
As shown in figure 51, several clones were found which bind equally to TEL and HEL in contrast to the original
D1 .3 scFv, which binds almost exclusively to HEL. None of the clones bound to BSA. These findings indicate that the
specificity of these scFvs was broader in comparison to D1 .3, since both lysozymes (HEL and TEL) are recognized,
but specificity for lysozyme was retained since other BSA was not recognized. The deduced amino acid sequences
30 (derived by DNA sequencing) of two light chains from clones MF1 and M21 , which correspond to clones 3 and 9 in
figure 51 are shown in figure 52.
In the case of isolated antibodies the experimental approach as described in this study may be particularly useful
if recognition of a wider range of different but closely related antigens is desired. For example, monoclonal antibodies
against viral antigens viral antigens like V3 loop of HIV-1 gp1 20 are in most cases quite specific for one particular virus
35 isolate because of the variability in this part of the HIV-1 env gene. The modification of such antibodies in the way
described in this example may lead to antibodies which cross react with a wider range of HIV-1 isolates, and would
therefore be of potentially higher therapeutic or diagnostic value.
A similar approach could be taken in which a light chain variable domain of desired properties is kept fixed and
combined with a library of heavy chain variable domains. Some heavy chains, for example VHD1.3 retain binding
40 activity as single domains. This may allow a strategy where VH domains are screened for binding activity when ex-
pressed on phage and then binding domains combined with a library, of VL domains for selection of suitable light chain
partners.
Example 47
45
Selection of a Phage Antibody Specificity by Binding to an Antigen attached to Magnetic Beads. Use of a Cleavable
Reagent to allow elution of Bound Phage under Mild Conditions
When a phage antibody binds to its antigen with high affinity or avidity it may not be possible to elute the phage
50 antibody from an affinity matrix with a molecule related to the antigen. Alternatively, there may be no suitable specific
eluting molecule that can be prepared in sufficiently high concentration. In these cases it is necessary to use an elution
method which is not specific to the antigen-antibody complex. Unfortunately, some of the non-specific elution methods
disrupt phage structure, for instance phage viability is reduced with time at pH12 (Rossomando, E.F. and Zinder, N.D.
J. Mol. Biol. 36 387-399 1 968). A method was therefore devised which allows elution of bound phage antibodies under
55 mild conditions (reduction of a dithiol group with dithiothreitol) which do not disrupt phage structure.
Target antigen was biotinylated using a cleavable biotinylation reagent. BSA conjugated with 2-phenyl-5-oxazolone
(O. Makela at al. supra) was modified using a biotinylation reagent with a cleavable dithiol group (sulphosuccinimidyl
2-(biotinamido) ethyl-1 ,3-dithiopropionate from Pierce) according to the manufacturers instructions. This biotinylated
74
EP0 774 511 A1
antigen was bound to streptavidin coated magnetic beads and the complex used to bind phage. Streptavidin coated
magnetic beads (Dynal) were precoated with antigen by mixing 650|ug of biotinylated OX-BSA in 1 ml PBS, with 200uJ
of beads for at least 1 hour at room temperature. Free antigen was removed by washing in PBS. One fortieth of the
complex (equivalent to 5uJ of beads and an input of 17.5 jag of OX-BSA) was added to 0.5ml of phage in PBSM (PBS
5 containing 2% skimmed milk powder) containing 1.9x10 10 phage particles mixed at the ratios of pAbD1.3 directed
against lysozyme (example 2) to pAbNQ11 directed against 2-phenyl-5-oxazolone (example 11) shown in Table 12.
After 1 hour of incubation with mixing at room temperature, magnetic beads were recovered using a Dynal MPC-
E magnetic desperation device. They were then washed in PBS containing 0.5% Tween 20, (3x10 minutes, 2x1 hour,
2x 10 minutes) and phage eluted by 5 minutes incubation in 50ul PBS containing 10mM dithiothreitol. The eluate was
10 used to infect TG1 cells and the resulting colonies probed with the oligo NQ11CDR3
( 5 ' AAACCAGGCCCCGTAATCATAGCC 3 ' )
15 derived from CDR3 of the NQ11 antibody (This hybridises to pAbNOH but not pAb D1 .3).
A 670 fold enrichment of pAbNQ11 (table 12) was achieved form a background of pAbD1.3 in a single round of
purification using the equivalent of 17.5|ag of biotinylated OX-BSA.
This elution procedure is just one example of an elution procedure under mild conditions. A particularly advanta-
geous method would be to introduce a nucleotide sequence encoding amino acids constituting a recognition site for
20 cleavage by a highly specific protease between the foreign gene inserted, in this instance a gene for an antibody
fragment, and the sequence of the remainder of gene III. Examples of such highly specific proteases are Factor X and
thrombin. After binding of the phage to an affinity matrix and elution to remove non-specific binding phage and weak
binding phage, the strongly bound phage would be removed by washing the column with protease under conditions
suitable for digestion at the cleavage site. This would cleave the antibody fragment from the phage particle eluting the
25 phage. These phage would be expected to be infective since the only protease site should be the one specifically
introduced. Strongly binding phage could then be recovered by infecting e.g. E.coli TG1 cells.
Example 48
30 Use of Cell Selection to provide an Enriched Pool of Antigen Specific Antibody Genes. Application to reducing the
Complexity of Repertoires of Antibody Fragment displayed on the Surface of Bacteriophage
There are approximately 10 14 different combinations of heavy and light chains derived from the spleen of an im-
munised mouse. If the random combinatorial approach is used to clone heavy and light chain fragments into a single
35 vector to display scFv, Fv or Fab fragments on phage, it is not a practical proposition to display all 10 14 combinations.
One approach, described in this example, to reducing the complexity is to clone genes only from antigen selected cells.
(An alternative approach, which copes with the complexity is the dual combinatorial library described in example 26).
The immune system uses the binding of antigen by surface immunoglobulin to select the population of cells that
respond to produce specific antibody. This approach of selecting antigen binding cells has been investigated to reduce
40 the number of combinatorial possibilities and so increase the chance of recovering the original combination of heavy
and light chains.
The immunological response to the hapten 4-hydroxy-3-nitrophenylacetic acid (NP) has been extensively studied.
Since the primary immune response to NP uses only a single light chain the applicants were able to examine the use
of the combinatorial method using a fixed light chain and a library of heavy chains to examine the frequencies genes
45 that code for antibodies binding to NIP (4-hydroxy-3-iodo-5-nitrophenylacetic acid). The applicants have thus used this
system to investigate the merits of selecting cell populations prior to making combinatorial libraries for display on phage.
Methods
50 2.1 Hapten conjugates
Chick Gamma globulin (CGG, Sigma, Poole, UK) and Bovine serum albumen (BSA, Boehringer, Mahnneim, Ger-
many) were conjugated with NP-O-succinimide or NIP-caproate-O-succinimide (Cambridge Research Biochemicals,
Northwich, UK) based on the method described by Brownstone (Brownstone, A., Mitchison, N.A. and Pitt-Rivers, R.,
55 Immunology 1 966. 1 0: 465-492). The activated compounds were dissolved in dimethylformamide and added to proteins
in 0.2 M sodium hydrogen carbonate. They were mixed with constant agitation for 16 hours at 4°C and then dialysed
against several changes of 0.2 M sodium hydrogen carbonate. They were finally dialysed into phosphate buffered
saline (PBS). The conjugates made were NP 12 CGG, NIP 10 BSA. The NIP 10 BSA derivative was subsequently bioti-
75
EP0 774 511 A1
nylated using a biotinylation kit purchased from Amersham (Amersham International, Amersham, UK).
2.2 Animals and immunisation
s Mice of the strain C57BL/6 were immunised by intraperitoneal injection of 100|ag NP-CGG in Complete Freunds
Adjuvant at 10 weeks of age.
2.3 Spleen preparation
10 Seven days after immunization cells from the spleen were prepared as described b.y Galfre and Milstein (Galfre,
G. and Milstein, C. Methods Enzymol. 1 981 . 73:3-46). Red cells were lysed with ammonium chloride (Boyle, W. Trans-
plantation 1968.6:71) and when cell selection was performed dead cells were removed by the method described by
von Boehmer and Shortman (von Boehmer, H. and Shortman, K, J. Immunol, Methods 1973:1:273). The cells were
suspended in phosphage buffered saline (PBS), 1% Bovine serum albumen, 0.01% sodium azide; throughout all cell
15 selection procedures the cells were kept at 4°C in this medium.
2.4 Cell Solution
Biotinylated Nl P-BSA was coupled to streptavidin coupled magnetic beads (Dynabeads M280 Streptavidin, Dynal,
20 Oslo, Norway) by incubating 10 8 beads with 100jag of biotinylated protein for 1 hour, with occasional agitation, and
then washing five times to remove unbound antigen. The coupled beads were stored at 4°C in medium until required.
For selection of antigen binding cells the cells (2-4x1 0 7 /ml) were first incubated for 30 minutes with uncoupled beads,
at a bead: cell ratio of 1:1, to examine the degree of non-specific binding. The beads were then separated by placing
the tube in a magnetic device (MPC-E Dynal) for 3-5 minutes. The unbound cells were removed and then incubated
25 with NIP-BSA coupled magnetic beads, at a bead:cell ratio of 0.1:1, for 60 minutes, with occasional agitation. The
beads and rosetted cells were separated as described above. The beads were then resuspended in 1 ml of medium
and the separation repeated; this process was repeated 5-7 times until no unbound cells could be detected when
counted on a haemocytometer.
For the depletion of surface immunoglobulin positive cells the cells were incubated with 20uxj biotinylated goat
30 anti-mouse polyvalent immunoglobulin (Sigma, Poole, UK). The cells were then washed twice with medium and added
to streptavidivin coupled magnetic beads at a bead to cell ratio of 30:1. After 30 minutes incubation the beads and
rosetted cells were separated by applying the magnetic device three times - taking the supernatant each time.
2.4 DNA/cDNA preparation, PCR amplification and cloning
35
DNA was prepared by a simple proteinase-K digest method that was particularly convenient for small numbers of
cells (PCR Protocols: A Guide to Methods and Applications. Ed Innis M.A., Gelfand D. H., Sninsky J.J. and White T.
J. Academic Press). RNA preparation and subsequent cDNA synthesis was performed as described by Gherardi at al
(Gherardi E., Pannell R. and Milstein C. J. Immunol. Methods, 1990. 126:61-68). PCR and cloning of the heavy chain
40 libraries was performed using the primers and conditions described by Ward at al (Ward, E.S., Gussow, D., Griffiths,
A.D., Jones, PT and Winter, G., Nature, 1989. 341: 544-546); 40 cycles of PCR amplification were performed. The
VH and Fv expression vectors used were adapted from those previously described by Ward at al. They were both
subcloned into pUC1 1 9 (Veira and Messing see later) and the Fv expression vector was modified to include a germline
lambda-1 light chain (obtained as a gift from T. Simon (originally cloned by Siegfried Weiss, Basel Institute of Immu-
45 nology)). THe vector is shown in Figure 53.
2.5 Expression and ELISA
For screening single colonies were picked into individual wells of microtitre plates (Bibby) in 200uJ 2 x TY/Ampicillin
so 100uxj/ml/0.1 % glucose and then incubated at 37°C for 5-6 hours with agitation, Isopropyl-p-D-thiogalactopyranoside
(IPTG, Sigma, Poole, UK) was then added to a final concentration of 1 mM and the incubation continued for a further
16 hours at 30°C before harvesting the supernatants. The wells of Falcon ELISA plates (Becton Dickenson, N. J.,
USA) were coated overnight at room temperature with NIP 10 -BSA (40|Lig/ml in PBS) and then blocked with 2% skimmed
milk powder in PBS for 2 hours at room temperature. The bacterial supernatants were added and incubated at room
55 temperature for 1 hour and then the plates were washed three times with PBS. Peroxidase conjugated-Goat anti-
mouse lambda-chain (Southern Biotechnology, Birmingham, USA) was added and again incubated for 1 hour at room
temperature before washing six times with PBS and then developing with 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sul-
fonic acid) (Sigma, Poole, UK) as the peroxidase substrate. The optical density at 405nm was measured using a
76
EP0 774 511 A1
Thermomax microplate reader (Molecular Devices, Menlo Park, USA) after 30 minutes. Western blotting using the C-
terminal myc tag as described in example 27.
3.1 Comparison of RNA/DNA and antigen selected cells
5
The results of antigen selection are shown in Table 13. Less than 1% of cells bind to NIP-BSA coated beads and
the non-specific binding is very low. Assessment of the proportion of expressed genes from each VH library using
western blotting showed that full length VH domains were expressed in 95% (1 9/20) of all clones when RNA was used
as the starting material but only 60% (1 2/20) of clones when DNA (either selected cells or from total spleen) was used
10 as the starting material. This difference probably results from the fact that many re-arranged pseudogenes could be
amplified with our primers and it appears that there must be some degree of selection, at the level of transcription, for
functional genes.
A variable number of clones from each type of library were screened for the production of Fv fragments that bound
to NIP. Initial screening ELISAs were performed and positives taken to include those with an optical density of at least
15 twice the background. The initial positives were retransformed and the binding checked in duplicate; it was confirmed
that the binding was specific to NIP and not to BSA. The frequency of confirmed positive NIP binding clones for each
starting material are shown in Table 14. Using DNA as the starting material for the PCR amplification is approximately
equivalent to sampling the cells present as there is only one functional re-arranged heavy chain gene and at most one
re-arranged pseudogene per B-cell. Amplifying from the RNA of an animal of course biases the repertoire to the reacting
20 B-cells and in a recently immunised animal this would be expected to give some bias towards the immunogen. The
data in Table 14 clearly shows how powerful this selection is with the number of antigen specific genes being enriched
at least 96 fold when RNA made one week after primary immunisation is used as the starting material. The data also
show that selection for antigen binding cells also provides an alternative powerful method of selection for the required
genetic starting material.
25
3.2 Comparison of Total Spleen/surface immunoglobuiin depleted Spleen
To examine the cellular basis of the selection achieved by using RNA as the starting material we depleted the
spleen of surface immunoglobulin positive cells using biotinylated anti-polyvalent immunoglobulin and streptavidin
30 conjugated magnetic beads. Prior FACS analysis had demonstrated that this method removed over 96% of surface
immunoglobulin positive cells. RNA was prepared from both surface immunoglobulin depleted and non-depleted fac-
tions of a spleen and VH libraries made from each. The ELISA results (Table 14) show that the number of positives is
certainly not decreased by this depletion suggesting that the major portion of the selective effect of using RNA may
come from surface immunoglobulin negative G-cells (probably plasma cells).
35
Conclusions
The applicants have demonstrated the importance of the amplification of specific RNA produced by immunisation
to enable binding activity to be obtained with any reasonable frequency from a combinatorial library. The applicants
40 have also demonstrated an alternative strategy which mimics that of the immune system itself. Using a simple method
of selecting for antigen binding cells gave comparable enrichment and has the added advantage of using a broader
range of genes. At first sight the random combinatorial approach would appear unlikely to produce the original com-
bination of heavy and light chain because of the vast diversity of the immunoglobulin genes. The applicants show here,
however, that following immunisation, with a good antigen, 1 0% of the VH genes from total splenic RNA isolated come
45 from antigen specific cells so the effective size of the repertoire is greatly reduced. This together with the fact that
promiscuity of the heavy and light chains occurs (examples 21 and 22) accounts for the fact that combinatorial system
does produce antigen binding clones with reasonable frequency. The data also suggests that the bulk of the antigen
specific RNA comes from surface immunoglobulin negative cells which are most likely plasma cells.
The data also show that this simple method of antigen selection may be useful in reducing the complexity of the
so combinatorial library. In this case an enrichment of antigen specific genes of at least 56 fold has been achieved which
in the normal case where heavy and light chains are unknown would result in a reduction of the complexity of the
combinatorial library by a factor of over 3000. A further advantage of using antigen selected cells (and amplifying from
DNA to reduce any bias due to the state of the cell) is that this results in a broader range of antibody genes amplified.
It may be that a simple cell selection such as that the applicants have described here in combination with phage
55 selection would be ideal. From this example it can be seen that by combining cell and phage selection methods one
could reasonably expect to screen all the combinations of heavy and light chain (approximately 4x1 0 10 ) and would
thus be able to screen all binding combinations although this would not, at present, be possible from whole spleen
(approximately 4x1 0 14 combinations, assuming 50% B-cells).
77
EP0 774 511 A1
Table 1.
Enrichment of pAb (D1 .3) from vector population
INPUT RATIO 3
OUTPUT RATIO
ENRICHMENT 01
oliao D
ELISA C
DAbfd-CAT1
nAb total ohaae
oAb total ohaae
Sinale Round
1:4x10 3
43/1 24
1.3x10 3
1:4x10 4
2/82
1.0x10 3
Two Rounds
1:4x10 4
1 97/372
2.1x10 4
1:4x1 0 5
90/356
3/24
1.0x10 5
1:4x10 6
27/1 83
5/26
5.9x1 0 5
1:4x10 7
1 3/278
1.8x10 6
Footnotes:
approximately 1 0 1 2 phage with the stated ratio of pAb (D1 .3) : FDTPS/Bs were applied to 1 ml lysozyme-sepharose columns, washed and eluted.
^TGI cells were infected with the eluted specific binding phage and plated onto TY-tet plates. After overnight incubation at 30-37°C, the plates were
analysed by hybridisation to the 32 p, labelled oligonucleotide VH1 FOR (Ward at al op cit) which is specific to pAb D1 .3.
c Single colonies from overnight plates were grown, phage purified, and tested for lysozyme binding.
^Enrichment was calculated from the oligonucleotide probing data.
Table 2
Enrichment of pAb (D1 .3) from mixed pAb population
Input Ratiol (pAbD1.3:pAbNQ11)
Output Ratio 2 (pAb D1.3:Total phage)
Enrichment
Single Round
1 : 2.5 x 10 4
1 8/460
0.98 x 10 3
1 : 2.5 x 10 5
3/770
0.97 x 10 3
1 : 2.5 x 10 6
0/112
pAb NQ11 only
0/460
Second Round
1 : 2.5 x 10 4
1 1 9/1 70
1.75 x 10 4
1 : 2.5 x 10 5
101/130
1.95 x 10 5
1 : 2.5 x 10 6
1 02/204
1.26 x 10 6
1 : 2.5 x 10 7
0/274
1 : 2.5 x 10 8
0/209
pAb NQ11 only
0/170
Notes
1 . 1 0 10 phage applied to a lysozyme column as in table 1 .
2. Plating of cells and probing with oligonucleotide as in table 1 , except the oligonucleotide was D1 .3CDR3A.
78
EP0 774 511 A1
Table 3:
Enzyme activity of phage-enzyme
Input
ng of enzyme or No. of phage
Rate (OD/hr)
No. of molecules of Enzyme equivalent
Pure Enzyme
i w< i w r i — i 1 1— y i i i \s
335
34
24 5
Pure Enzvme
177 5
lit.
17 4
i * ■ i
12 25
! Pure Enzvme
1 1 w 1— 1 1 Z_ y 1 1 1 w
88 7
8 7
6 1 25
Purp Fnzvmp
i ui u i_ i i z_ y i i
44 4
4 12
3 06
! Pure Enzyme
22.2
1.8
1.5
: Pure Enzyme
11.1
0.86
0.76
, No Enzyme
0
0.005
0
fd-phoAla166/TG1
1.83X10 11
5.82
4.2
fd-CAT2/TG 1
1.0x10 12
0.155
0.112
fd-phoAla166/KS272
7.1x10 10
10.32
7.35
fd-CAT2/KS272
8.2x10 12
0.038
0.027
79
EP0 774 511 A1
10
15
20
25
30
O
-e
o<
o
OX
a
.5
S3
g
35
40
45
50
0>
OX)
CQ
a<
WD
g
c
i
a
+■*
sa-
cs
o
c
55
H
u
§
13
C
2
c
e
o
en
oo
oo
IT)
CO
00
u
.2
o
s
o
U
s
o
■a
oo
oo
CO
■a
2
u
u
0)
S5
pq
On
00
CO
00
co
ST
i
>
a
>
o
oo
co
co
*R
co o,
£ £
S3
i
>
ST
r
X
>
CO
ON
ON
ON
<N
vB
ON
<N
ON
ON
v — '
<N
ON
> — '
On
*
vq »o
* — <» ^ — '
VO o
On tJ-
oo r-
00 T?
OO T*
13 W
W3
o
7j
o
D
3
x
•a
o
o
o
>
i
as
>
0>
o
in
1
3
U
CU
o
00
§
C»
Cu
un
cd
O
u
CI.
a
O
o
* i
■ ^^^^
o>
o g
« °
o o
H §
BO o>
■a a
o ^
Q «
^> 2
si
o >
,0 .^H
6 O
2 C
80
EP0 774 511 A1
Table 5
Soluble
chain(s)*
heavy chain
neavy cnain
coo
>«oo
rjL*
to
heavy chain
heavy chain
light chain
lignt chain
Chain as
gene III fusion*
scFv
light chain
scFv
light chain
heavy chain
light chain
light chain
heavy chain
Chain(s)
displayed*
none
scFv
Fab
none
scFv
Fab
heavy chain
light chain
none
Fab
none
Fab
Binding to
phOx*
non binding
binding
binding
non binding
binding
binding
binding
binding
non binding
non binding
non binding
binding
non binding
binding
Helper Phage
VCSMB
VCSMB
VCSMB
VCSMB
fd-tet-DOGl IV
VCSMB
fd-tet-DOGl III
4—
-a
s
CD
W)
cd
43
1 8i
cd
43
Oh
fd CAT2
fd CAT2-I
fd CAT2-II
pHENl
pHENl I
pHENl II
pHENl I (HB2151)
pHENl II (HB2151)
fd CAT2-III
fd CAT2-IV
pHENl III (HB2151)
pHENl III (HB2151)
pHENl IV (HB2151)
pHENl IV (HB2151)
<
oo
a
o
»
2
or?
t3
O
O
s
bO
cd
43
■a
cd
cd
3>
cl
o
oo
<
<
00
I
o
•Si
&
O
cd
<D
■*— »
cd
a)
bO
2
oo
cd
cd
CO
cd
Oh
e
cd
CO
o
Q
o
■s
-a
Si
."2
"oo
53
O
O
g
CD
&
CO
CO
cd
CD
CO
CO
CO
CD
•a
cd
bO
cd
43
CL,
CD
•3
2
bo
CD
rS
a
CO
CO
cd
8
oo
si
C3
2
oo
1
CD
CD
a
•— <
CO
ah
1
,o
<+-<
3
O
■4— i
CO
O
e
CD
ne
■«— >
CO
CD
cu
bJO
s
O
43
cd
1
o
CD
CD
CD
a
ex.
# o
%-»
CD
cd
CD
.O
a 1
* 8
T3
CD
+->
cd
o
J3
cd
o
• «— «
CD
CD
CO
CO
as
•»-»
to
oo
&
too
cd
cd
*0
bO
C3
1
CD
o
C3
cd
O
o
o
cd
81
EP0 774 511 A1
Table 6.
Kinetic parameters of soluble and phage-bound alkaline phosphatase. Relative values of k cat and K m for the
soluble enzyme and for the phage enzyme were derived by comparing with the values for wild type enzyme
(phoArg166) and the phage-wild type enzyme (fdphoArg166).
Soluble enzyme
Phage enzyme
(Data from et al
Chaidaroglu
(Data from this study)
1988)
phoArg166
phoAla166
phoArg166
phoAla166
K m (jllM)
12.7
1620
73
1070
Relative K m
1
127
1
14.6
Relative k cat
1
0.397
1
0.360
Relative k cat /K m
1
0.0032
1
0.024
Table 7
Enzyme Activity of Phage Samples
SAMPLE (Construct: host)
INPUT PHAGE
PARTICLE (pmol:)
RATE (pmol substrate
converted/min)
SPECIFICACTIVITY(mol
substrate converted/mol
phage/min)
fdphoArg166:TG1
2.3
8695
3700
fdphoAla166:TG1
5.6
2111
380
fdphoAla166:KS272
1.8
2505
1400
fdCAT2:TG1
3.3
<1
<0.3
fdCAT2:KS272
5.6
70
12
Table 8.
Affinity chromatography of phage-enzymes
SAMPLE
INFECTIVITY (Percentage of
phage particles which are
infectious)
INPUT PHAGE PARTICLE (x
10 9 )
OUTPUT PHAGE PARTICLE
(x 1 0 9 )
fdphoArg166
0.37%
5160
30
fdphoAla166
0.26%
3040
90
fdCAT2
4.75%
4000
2
45 Table 9
Mutations in scFvBI 8 selected by display on phage following growth in mutator strains
Nucleotide mutation (base position)
Amino acid mutation
Number
308
Ala^Val (VH FR3)
3
703
Tyr^Asp (VL CDR3)
1
706
Ser^ Gly (VL CDR3)
1
724
Gly^ Ser (VL FR4)
21
725
Gly-^ Asp (VL FR4)
3
734
Thr^lle (VL FR4)
1
82
EP0 774 511 A1
CD CD
o cd
cj y
CD Q
cj o
o o
H O
o <
cd u
55
o u
o o
o o
< <
u u
u u
O O
1~ CO
CO i
CD O
< <
U U
O O
£2
CD O
H <
O U
o o o o
H H H H
u u o u
u u
o cd
H H
O O
CD CD
< <
o u
< <
u u
o <
H H
O CD
O O
o u
wo
wo
wo
WO
WO
WO
M
M
U
U
U
u
U
U
<
<
<
<
<
<
PQ
PQ
PQ
PQ
3
cd
cd
cd
cd
cd
CO
Tf
wo
a
a
a
a
>
>
>
>
>
>
B
a
a
a
a
a
CO CO CO CO
666o
o cd o o
H H H CD
H H H H
cd o o o
< < < <
u o cd o
CO
CO
o
cd
H
U
<
o u
o o
o a
< <
u u
u u
u y
a a
CD CD
U CJ
CD CD
a a
u u
u u
o a
< <
u u
u u
u y
a cd
a cd
u w
CD CD
^^^^ ^^^^
u cj
< <
< <
u u
u u
CJ CJ
H H
a o
8
CD CD
H <
o u
p a
U CJ
CD CD
< <
u u
p CD
o a
a cd
Is <
CD U
u a
CJ CJ
a a
CD CD
H H
< <
u u
CD O
o o
CJ CJ
u u
S3
u u
u u
CJ CJ
CD CD
CD O
cj y
CD CD
u cj
< <
< <
cj cj
O CD
a o
H <
u P
3 CJ
CD O
H <
H CJ
CD CD
E— H
CJ CJ
S3
U CJ
o <
H H
O CD
o o
< <
u u
u o
y cj
a o
CD CD
< s
gg
CD CD
CJ CJ
u u
o o
< <
u u
o u
CJ CJ
O CD
CD CD
CJ CJ
CD CD
H H
U CJ
< <
< <
CJ CJ
a cd
uuuu
^H^^ ^^B^^ ^^W^^ ^^B^^j
uuuu
uuuu
H H H b«
CD CD CD CD
wo wo wo wo wo wo
in
is
is
in
00
oo
00
u
U
U
U
U
U
<
<
<
<
<
<
PQ
PQ
PQ
PQ
PQ
a
cd
cd
cd
CN
CO
wo
a
a
a
a
a
>
>
>
>
>
>
a
a
a
a
a
a
CO
Vh
s
o
PL,
a
6
CO
1
*>
CO
CO
1
CO
1
U
1
U
U
U
U
U
U
CD
U
U
H
CD
CD
O
CD
H
CD
CD
CD
H
O
<
<
<
a
u
U
U
CJ
CJ
U
CJ
CJ
g
s
<
<
CD
CD
H
H
H
H
O
O
O
a
O
o
O
CD
CJ
CJ
CJ
CJ
<
<
<
<
CD
CD
CD
CD
<
<
<
<
a
CD
o
<
a
CD
<
<
<
<
o
o
CD
CD
H
H
I
H
1
H
1
wo
•>
WO
wo
WO
o
p^
O &
TJ- SO
a a
^)
d ^ p 3
a a a a
o
CO
83
EP0 774 511 A1
CO CO
O
r-H
■* o
o S
CO
0)
a
o
cd
m
>
s
< <
O o
< < H
O o
< H
U O
H H
< O
U H
CO CO
I I
o u
u u
o p
O O
< <
u v
o o
o u
< <
o o
<
o a
H H
O O
< <
CO "
I CO
(J
u
H
U
O
<
(J
U
o
S
<
u
< o
< <
< u < <
< < < <
o o o o
v) m in in io
a u
< <
t-H CN
> >
us w
< <
> >
US M
O O
< <
PQ PQ
> >
00
CD
£
o
co co co
DUO
U O U
u o u
co co
u u
u u
u u
E- H H H H
O O O O O
a o o o h
- r, - ^ 8
O O H U H
u O U O o
< < < < <
u u u u u
u o u
H H H
H U <
H H H
< < <
O O O
O O O
O U U
< < <
«o u-) <n >o >r»
OS
O O
i — ( cN
« PS
o o o
(Xi
co Tt* *n
^ Vi K
9 3 9
ffi ffi ffi
CO
1
C*>
CM
CJ
1
1
U
r \
i
1
r \
vJ
r \
\J
r \
r \
w
r \
r \
\J
rj
o
rj
rj
CJ
H
r .
r -1
o
ro
rn
c
r
h
H
o
H
u
H
u
rj
rj
L
U
o
U
U
o
<•
<<<
u
o
U
u
u
o
H
H
r j
r )
r .
r j
<*
r ,
r ,
P
r ,
r- 1
P
H
o
CD
rj
<
<
<
<
r \
r )
r )
T )
r >
rn
<J
o
u
(J
(J
(J
r )
r j
ro
ro
O
o
CD
u
o
U
U
rn
rn
rn
H
H
H
H
H
r .
b H
r ,
r .
r \
r \
vJ
vJ
<<*
^«
r W
o
O
o
o
u
o
o
U
u
H
H
H
H
H
u
u
u
u
o
<
<
<
<
<
u
o
u
u
H
t-
H
H
O
o
O
O
a
<
<
o
1
o
1
S
1
1
i
«o
*>
o
< <
—i <S
B 33
u u u
<<<
PQ CQ PQ
co tj- in
v v: id
3 9 3
33 33 W
84
EP0 774 511 A1
co
i
u
O
< S
H P.
O o
U O
o h
a h
u u
a p
< P
H O
?n H <
Sao
U <J u
H U O
POO
o o o
< u u
< o o
O H H
H H H
H <J U
O < <
H O O
uuu
U H H
U U <J
U H H
H H H
U < <
H U U
U H H
< o o
o < <
< o o
I I I
in in
o o
O O O
Ph Uh Ph
* *i
p p a
S3 33 33
on
CD
a
a
PQ
a
s
cn en
U U
u u
< u
u <
u o
CO
u u
u o
O H
u u
u u
o o a o
< < < <
u u u u
uuuu
< < < <
o o
F H
H U
o u
H U
O O
H H
U U
H H
a o
< <
u u
CO CO cO
U U <
o o o
8 u 8
u u <j
H H h
U U 5
< < <
o o o y o
H H H H H
U U U U (j
<go
H H h
< O <
h <:
o o
H H
< U
H H
U H
U U
H H
h y p
O O p
U O H
< < <
U U <
>o io »n «n «o <o in
U U
< <
PQ pq
> >
P P
33 33
U U
< <
co PQ
CO CO
> >
p p
33 ffi
www
uuu
< < <
PQ PQ PQ
\r> \o
r< ^ r<
> > >
DPP
CO
Ph
O
B
CO CO ^
o u o
U O P
u u o
f-^
o o o
o o o
hHp
H H H
UUU
U O O
^ < ^
o u o
H H H
OOO
OOO
UUU
< < <
a o <
o o <
< < <
H H H
UUU
UUU
< < <
t 1 t
«o *o *o
Pi p^
Pi o o
o *
Ph *?
s< s< s<
"3 *3 ^3
ffi K S
CO
CO
CO
1
u
1
u
U
U
U
U
U
U
r >
H
H
E- 1
O
O
U
r ^
ro
H
O
H
H
r ,
r \
r ^
U
O
<
<
r \
rh
H
H
H
O
U
ro
U
rO
ro
U
U
U
<
<
<
U
_ * —
0
<
<
r
H
H
U
U
>^
U
U
U
<
<
<
u
u
U
0
0
O
u
u
u
u
u
u
0
0
0
0
0
0
rj
0
0
0
H
H
H
H
H
H
u
u
U
<,
<
O
u
u
u
H
H
H
U
U
P
U
p
<
<
U
u
u
r_ .
r,
M
O
O
O
<
<
<
1
1
ro
1
H
O
O
H
O
O
O
O
CO
1
wo
1
PL,
<-<
^<
S
85
EP0 774 511 A1
10
15
20
25
30
35
40
45
50
55
3
H
m
i
H
H
U
g
u
<:
o
o
co
I
H
H
L_ U
b °
u o
< H
o o
o
co
h < ^
H H O
u ^
o o o
H u U
y CJ U
< o o
<-> o o
^ u o
O o o
O H H
o u u
< < <
H O O
O U U
H H H
tee
< < <
o u u
< H H
< o o
o < <
H O O
I I I
Ik «k «.
m *o m
^ ^3
o o
o o o
(i. PL, pl,
^
U U U
P Z3 P
K E ffi
00
o
t>0
>
o
co co
i •
O O
o o
H H
O O
O O
< <
H H
U H
U o
H H
O O
U <->
O U
< <
o p
U <
U <
o u
i i
o u
a o
a o
a o
< <
u u
a u
O P
a o
u o
u u
< <
u p
o o
o o
o <
u u
u u
< < < <
u o < o
o o o o
till
*» #*. M
<o »n >o >o
i-H m Tt
PL,
a.
U
i
3
<u
>
CO
o
PL.
o
3
>
o
DO
o
>
DO
>
a>
OS
en to co ro
0 u
o <->
o o
u u
a o
u u
u u
a <
<: <
a <
o u
o u
< <
a o
a <
o o
U U
U U
O O
O U
V U
H H
o o
u p
H H
H O
P u
<
O U
H H
o o
u o
o o
o u
U ^
< <
o o
u ^
o P
H H
H
H
O
a
H
O
O O
< <
H H
o a
u <
a a
u u u u u u
<<<<<<
o o o o o o
<<<<<<
o o o o o o
0 o o o o o
1 I I I I I
in «n in in in
>>>>>>
LL LL LL (L LL Li
U u J U J tJ
HH hh hh
O U U V u o
<<<<<<
CD ffl CQ CQ CQ ffi
cxj (Xl aj aj 03 cC
— * <N m Tj- in
^ « « « «
>>>>>>
3 g 3 3 3 ^
>
oo
Ui
>
Pi
86
EP0 774 511 A1
fn
\* i
1
" 1
i
o
i
o
1
ro
CO
ro
co
H
H
H
H
r \
CJ
\J
co
CO
CO
CO
U
CJ
o
o
r .
r ,
<
<f
<
u
cj
CJ
r \
W
CJ
vj
CO
O
CO
CO
. . *
— ■ k_
o
o
o
o
CJ
u
cj
L
L '
o
H
■r L.
o
r )
f— <
<
<
<:
<
O
<
O
o
U
H
<;
<
<
U
u
u
H
<
<
<
<
<
V
CJ
u
U
H
H
H
H
O
O
o
O
O
u
o
O
O
o
o
O
H
H
H
U
u
U
U
<
<
<
<
O
<
<
<
<:
o
o
o
o
o
t
o
1
o
1
o
i
10
*/~>
< <
O O
< <
o u
< <
< <
M
< <
PQ PQ
> >
3 3
u
PQ
<
m
03 as
o u
< <
PQ PQ
> >
■a
>
>
CO
>
>
a;
*? *? *? co *V *°
0900606
< ^ < < < < <
o o o o o o o
< < < <
u u u o
u o u u
o o
u u
u u
< <
u u
o u
a o
u u
U CJ
H H
< ^
a o
u y
o p
H H
O U
< ^
< <
u y
< o
o <
< o
< <
u p
O O
u <
0 a
u u
a o
o o
y <
o u
a a
< < <
u p o
U CJ u
o o o o o
p u u U u
o u u U u
< <
u u
o o
o o
P CJ
CJ u
h H ^ W
<c < < < <
o o a cd o
y y u y u
< < O O H
O O H H H
^ S 5
< < <
CJ p CJ
O CJ o
o p o
cj p u
CJ CJ o
o
5
0
CJ u
< H
y u
< <
P CJ
a o
p 0
y cj
o u
O H
H O
o o
a a
< < <
H CJ H
< < <
H CJ U
000
< < <
u p u
H H H
000
< < <
000
H H H
H U U
000
000
0 o o
o < o
O O H
1 I I
*0 lO lO tr> *0 10
> >
lift
2 « M
u CJ CJ
2 < <
3 S 9
> > >
3 3
> >
u u
< <
CO CO
> >
a 3
87
EP0 774 511 A1
10
15
20
25
30
35
40
45
50
S S S < c-
~ 3
o\ U A o
" *~ 9
CO < 01 ►-« •—
jcs 3 3 a
x> *a a cn
(5 05 a th
cn cn 05 o
cn 2 r cn
o o ^ n
^ < cn cn
§835
5555
° O Q Q
5s££
SSgS
^ ^ ^ ^
♦< ffl K K
2§29
O Cn
333
noon
5 5 in 8
3 3 § -
cn cn co
CO 73 T\
= ~ = 5 5
tr» cj o
Q
cn
a
*<
cn cd co cn
"«] *n p m
cn cd cn cn
Z *t cn
«< 03 O
a a
CO
a a
O O JO »
-4 H O O
> > *»J *n
S
v:
3
a
w O
Q
cn 5
CO »-»
PS
cn
g
cn
cn
Q
s
Co
>
O —
o ?
o ~
r cn
<
o
CD
-4
cn
cn
CL
CO
< r
~ o
a
3
CQ
3:
•<
o
>
•a
I
Cn
Q
«— >
o
**)
cn
Q
cn
»— ♦
s
a
cn
<4
:>
e
8
-a
s
c
=3
or
— «
r-
a
m
a
c
CI
O
a
<
0Q_
O
S3
■
3
tn
(n
a
a
rt
a
a
55
3
3
<
88
EP0 774 511 A1
Table 12
Enrichment of pAbNQ11 from pAbD1.3 background by affinity selection using Ox-BSA biotinylated with a
cleavable reagent and binding to streptavidin magnetic beads
Input Ratio 1 (pAbD1 .3:pAbNQ1 1 )
Output Ratio 2 (pAb NQ11: Total phage)
Enrichment
2235:1
61/197
690
22350:1
5/202
544
1 . 1 .9x10 lu phage in 0.5ml mixed for 1 hour with 5uJ streptavidin-magnetic beads precoated with antigen (OX-BSA).
2. Colonies probed with the oligonucleotide NQ11CDR3
Table 13:
Results of antigenic cell selection
Number of Cells
% of total cells
Total spleen cells
4x1 0 7
Cells bound to uncoated beads
0.8x1 0 4
0.02
Cells bound to NIP-BSA coated beads
22x1 0 4
0.55
Table 14:
Results of Fv NIP binding ELISAs from selected cell populations:
Positives
*Degree of Enrichment
Cell Population
DNA from total spleen
0/940
RNA from total Spleen
29/282
> 96
DNA from antigen binding cells
1 7/282
>56
Surface Ig Selection
RNA from Surface Ig negative fraction
8/94
RNA from total Spleen
4/94
* Degree of enrichment compared to total DNA.
35
Claims
1. A method of producing a member of a specific binding pair (sbp), which method comprises:
expressing in recombinant host cells nucleic acid encoding said sbp member or a genetically diverse population
of that type of sbp member, wherein the or each said sbp member or a polypeptide component thereof is expressed
as a fusion with a surface component of a secreted bacteriophage which displays at the surface of the bacteri-
ophage particle said sbp member in a functional form comprising a binding domain for a complementary sbp
member, said particle having the ability to replicate provided by genetic information packaged therewithin using
said surface component, nucleic acid encoding said displayed sbp member or a polypeptide component thereof
being contained within the host cell in a form that is capable of being packaged in said particle using said surface
component, whereby the genetic material of the particle displaying an sbp member encodes said displayed sbp
member or a polypeptide component thereof.
2. A method according to claim 1 wherein said displayed sbp member is multimeric and said polypeptide component
is a first polypeptide chain of said displayed sbp member, the method further comprising expressing in a recom-
binant host organism a second polypeptide chain of said multimer and causing or allowing the polypeptide chains
to come together to form said multimer as part of said particle, at least one of said polypeptide chains being
expressed from nucleic acid that is capable of being packaged in said particle using said surface component,
whereby the genetic material of each said particle encodes a polypeptide chain.
3. A method according to claim 2 wherein both said chains are expressed in the same host organism.
89
EP0 774 511 A1
4. A method according to claim 3 which comprises introducing a vector capable of expressing said first polypeptide
chain into a host organism which expresses said second polypeptide chain in free form, or introducing a vector
capable of expressing said second polypeptide in free form into a host organism which expresses said first polypep-
tide chain.
5. A method according to claim 3 wherein said first and second chains of said multimer are expressed as separate
chains from a single vector containing their respective nucleic acid.
6. A method according to any one of the preceding claims wherein the displayed sbp member or a said polypeptide
component thereof is expressed from a phage vector.
7. A method according to any one of claims 2 to 6 wherein each said polypeptide chain is expressed from nucleic
acid which is capable of being packaged in a said particle using said component fusion, whereby encoding nucleic
acid for both said polypeptide chains are packaged in respective particles.
8. A method according to any one of the preceding claims wherein the nucleic acid encoding the or each displayed
sbp member or a said polypeptide component thereof is obtained from a library of nucleic acid including nucleic
acid encoding said chain or a population of variants of said chain.
9. A method according to claim 8 wherein the displayed sbp member is a multimer comprising first and second
polypeptide chains and both first and second polypeptide chains are obtained from respective said libraries of
nucleic acid.
10. A method according to any one of claims 1 to 9 wherein the or each displayed sbp member or a said polypeptide
component thereof is expressed from a phagemid vector, the method including using a helper phage, or a plasmid
expressing complementing phage genes, to help package said phagemid genome, and said surface component
is a capsid protein therefor.
11. A method according to claim 10 wherein said capsid protein is absent, defective or conditionally defective in the
helper phage.
12. A method according to claim 11 wherein said helper phage is obtainable from recombinant E. coli TG1 M13K07
gill No.3, deposited as NCTC 12478.
13. A method according to claim 1 to 9 wherein said fusion is with bacteriophage capsid protein and the particle is
formed with said fusion in the absence of said capsid protein expressed in wild-type form.
14. A method according to any one of claims 1 to 9 wherein said fusion is with a bacteriophage capsid protein and a
native said capsid protein is present in said particle displaying a said fusion.
15. A method according to claim 14 wherein a single said fusion is displayed per particle displaying a said fusion.
1 6. A method according to any one of the preceding claims wherein the or each displayed sbp member or a polypeptide
component thereof is expressed in a host cell which is a mutator strain which introduces genetic diversity into the
sbp member nucleic acid.
17. A method according to any of claims 1 to 15 wherein said genetically diverse population is obtained by in vitro
mutagenesis of nucleic acid encoding a sbp member or polypeptide component thereof.
18. A method according to any one of claims 1 to 16 wherein genetically diverse population is obtained from:
(i) the repertoire of rearranged immunoglobulin genes of an animal immunised with complementary sbp mem-
ber,
(ii) the repertoire of rearranged immunoglobulin genes of an animal not immunised with complementary sbp
member,
(iii) a repertoire of an artificially rearranged immunoglobulin gene or genes,
(iv) a repertoire of an immunoglobulin homolog gene or genes, or
(v) in vitro mutagenesis of nucleic acid encoding an immunoglobulin gene or a library of immunoglobulin genes,
90
EP0 774 511 A1
or
(vi) a mixture of any of (i), (ii), (iii), (iv) and (v).
19. A method according to any one of the preceding claims wherein said displayed sbp member comprises a domain
s which is, or is homologous to, an immunoglobulin domain.
20. A method according to any one of the preceding claims wherein the host is a bacterium and said surface component
is a capsid protein for the bacteriophage.
10 21. A method according to claim 20 wherein the bacteriophage is a filamentous phage.
22. A method according to claim 21 wherein the phage is selected from the class I phages fd, M1 3, f 1 , If 1 , Ike, ZJ/Z,
Ff and the class II phages Xf, Pf 1 and Pf3.
15 23. A method according to claim 21 or claim 22 wherein the or each displayed sbp member or polypeptide component
thereof is expressed as a fusion with the gene III capsid protein of phage fdor its counterpart in another filamentous
phage.
24. A method according to claim 22 wherein said displayed sbp member or polypeptide component thereof is inserted
20 in the N-terminal region of the mature capsid protein downstream of a secretory leader peptide.
25. A method according to any one of claims 20 to 24 wherein the host is E.coli.
26. A method according to any one of the preceding claims wherein nucleic acid encoding an displayed sbp member
25 polypeptide is linked downstream to a viral capsid protein through a suppressible translational stop codon.
27. A method according to anyone of the preceding claims wherein the particles formed by said expression are selected
or screened to provide an individual displayed sbp member or a mixed population of said displayed sbp members
associated in their respective particles with nucleic acid encoding said displayed sbp member or a polypeptide
30 component thereof.
28. A method according to claim 27 wherein the particles are selected by affinity with a member complementary to
said displayed sbp member.
35 29. A method according to claim 28 which comprises recovering any particles bound to said complementary member
by washing with an eluant.
30. A method according to claim 29 wherein the eluant contains a molecule which competes with said particles for
binding to the complementary sbp member.
40
31. A method according to any one of the claims 28 to 30 wherein the particles are applied to said complementary
sbp member in the presence of a molecule which competes with said particles for binding to said complementary
sbp member.
45 32. A method according to claim any one of claims 27 to 31 wherein said sbp member comprises or sbp members
comprise a binding domain formed by an antibody light chain variable domain and an antibody heavy chain variable
domain, and an antibody heavy or light chain variable domain of an sbp member provided by a selected or screened
particle is combined with a library of polypeptides comprising respective complementary light or heavy chain an-
tibody variable domains and sbp members able to bind a complementary sbp member are selected.
50
33. A method according to any one of claims 27 to 31 , wherein nucleic acid derived from a selected or screened particle
is used to express said sbp member which was displayed or a fragment or derivative thereof in a recombinant host
organism.
55 34. A method according to claim 32 or claim 33 wherein nucleic acid from one or more particles is taken and used to
provide encoding nucleic acid in a further method to obtain an individual sbp member or a mixed population of sbp
members, or encoding nucleic acid therefor.
91
EP0 774 511 A1
35. A method according to claim 33 or claim 34 wherein the expression end product is modified to produce a derivative
thereof.
36. A method according to any one of claims 33 to 35 wherein the expression end product or derivative thereof is used
to prepare a therapeutic or prophylactic medicament or a diagnostic product.
37. Recombinant host cells harbouring a library of nucleic acid fragments comprising fragments encoding a genetically
diverse population of a type of member of a specific binding pair (sbp), each sbp member or a polypeptide com-
ponent thereof being expressed as a fusion with a surface component of a secretable bacteriophage, so that said
sbp members are displayed on surface of bacteriophage particles in functional form comprising a binding domain
for a complementary sbp member and the genetic material of the particles, packaged using said surface compo-
nent, encodes the associated displayed sbp member or a polypeptide component thereof.
38. Recombinant host cells according to claim 37, wherein said type of displayed sbp member are immunoglobulins
or immunoglobulin homologs, a first polypeptide chain of which is expressed as a said fusion with a surface com-
ponent of the particle and a second polypeptide chain of which is expressed in free form and associates with the
fused first polypeptide chain in the particle.
39. A secreted bacteriophage displaying in functional form on its surface a member of a specific binding pair (sbp)
comprising a binding domain for complementary sbp member.
40. A bacteriophage according to claim 39 wherein the displayed sbp member is multimeric.
41 . A bacteriophage according to claim 39 or claim 40 wherein the displayed sbp member comprises a binding domain
of an immunoglobulin.
42. A bacteriophage according to any one of claims 39 to 41 which is a filamentous phage and said displayed sbp
member is displayed as a fusion with a capsid protein of the phage, the phage displaying a single said fusion.
43. A bacteriophage according to any one of claims 39 to 40 which is a filamentous phage and said displayed sbp
member is displayed as a fusion with a capsid protein of the phage, all said capsid proteins of the phage being
expressed as fusions with said displayed sbp member, the phage genome being a phagemid not containing a
complete wild-type phage genome.
44. A bacteriophage according to any one of claims 39 to 43 which is a filamentous phage and said displayed sbp
member is displayed as a fusion with a capsid protein of the phage which is gene III of phage fd or its counterpart
in another filamentous phage.
45. A kit for use in carrying out a method according to any one of claims 1 to 36, said kit including:
(i) at least one vector having an origin of replication for single-stranded bacteriophage, nucleic acid encoding
said sbp member or a polypeptide component thereof in the 5' end region of the mature coding sequence of
a phage capsid protein, and with a secretory leader sequence upstream of said site which directs a fusion of
the capsid protein and sbp polypeptide to the periplasmic space of a bacterial host; and
(ii) ancillary components required for carrying out the method.
92
EP0 774 511 A1
EP0 774 511 A1
Fig. 2 ft)
Fig. 2( ii )
p
O Oc
t
ag
Q
ag
94
EP0 774 511 A1
tet A
fd- tet
V
V
clGave with BsfEII
V
V
fill in with KlGnow
V
V
re-ligate
f
FDT6 Bst
V
in vitro mutagenesis (oligo 1)
T
FDTPs/Bs
V
V
in vitro mutagenesis (oligo 2)
T
FOTPs/Xh
95
EP0 774 511 A1
LJ
LD
<r
ld
LJ
LJ
<
ID
ID
£
ID
<
LJ
ID
LJ
lj
LD
LD
LJ
LJ
<
LD
I —
LD
LD
LJ
<
LD
<
LD
LD
<
LD
CM
NO
LD
LJ <
p LD
h- LD
I — p h—
<t LD
m
LTl <£ <£
vO l_j LD
^ < ID
LD
LJ
LJ
LD
LD
<C
LJ
LD
LJ
LJ
LD
<
LD
LJ
<
LD
LD
LJ
LJ
8
<c
LJ
LJ
LTI <T
sO LJ
i_n LD
vo <C
LD
LD
LD
LJ
<
<
<
LD
LD
LD
LJ
<£
LD
<
LJ
LJ
LJ
O LJ
^3 LD
LU
oo
< ID
00 <
uu
LJ
LJ
LD
LJ
LJ
h—
LJ
<
_ LJ
vO LJ
LD
m
o
o
o
cn
cn
* « *
o
O
O
LD
<
<
LD
LD
LJ
„ <
00 LJ
f—
LJ
0O LJ
LJ
h—
LD
LJ
LJ
<c
LJ
h~
LD
LD
LJ
<
LJ
LD
<
<
LD
C3 <
LJ
—J
LJ
a <
lj
O LD
LD
LJ
LJ
<
LJ
I —
LJ
GO
CD
CL
LD
<
<
LD
LD
h-
LJ
<
<
<
LD
LD
CH LD
LJ
<
LJ
LD O
lu < x:
LD X
LJ
LJ
LD
<
LD
LD
<
LJ
LD
— 1 I —
LJ
<
a <
lj
LJ
> f —
LD
_ LD
LJ
LJ
LD
LJ
LJ
LJ
<
LJ
LJ
LJ
CO
Ql
LL
96
EP0 774 511 A1
Fig. 5.
rbs MKYLLPTAA
GOSECAAATTCTATTIX^^
10 20 30 40 50 60
SphI
PelB leader
AGLLLLAAOPAMA Q V Q L Q E S
70 80 90 100 110 120
PstI
GPQLVAPSQSLSITCTVSGF
GGACCIXSGCCIXjGTGGCGCCCTCA
130 140 150 160 170 180
S LTGYGVNWVRQ P PG KGLEW
TCATTAACXX3GCTATCG^
190 200 210 220 230 240
VHD1.3
LGMIWGDGNTDYNSAL KSRL
CIX3GGAATGATrTOGGGTGAT^^
250 260 270 280 290 300
S ISKDNS. KSQVFLKMNSLHT
AGCATCAGCAAGGACAACTTCC^
310 320 330 340 350 360
DDTARYYCARERDYRLDYWG
GATGACACAGCCAGGTACTAC^
370 380 390 400 410 420
Linker Peptide
QG.TTVTVSS GGGGSG GGGSG
aU\GGCACCACQffI^
43D 440 450 460 470 480
BstEII
TQS PASLSASVG
CTCAGTCTCCAGCCT^
510 520 530 540
Q Q Q S D I E L
ggt ggcgga t cgGACATCGAGCE
490 500
Sac I
97
EP0 774 511 A1
Fig. 5 con t
ETVTITC RASGN I HNYLAWY
GAAACIXjTCACCATCACAT^
550 560 570 580 590 600
QQKQGKS PQLLVYYTTTLAD
CAGCAGAAACAGGGAAAATCT^
610 620 630 640 650 660
VKD1.3
GVPSRFSGSGSGTQYSLKIN
GGrKTIKXrCATCAAGGT^
670 680 690 700 710 720
SLQPEDFGSYYCQHFWSTPR
AGCCTGCAACCTGAAGATTTIT^
730 740 750 760 770 780
Myc Tag (TAQl)
TFGGGTKLEI KR EOKLISEE
ACGflTCGGTDGGAGGGACCAA^
790 800 810 820 830 840
Xhol
D L N * *
GATCTGAATTAATAATGA
850 860 870 880
EcoRI
98
EP0 774 511 A1
O.D.
405 nm
2n
1-
0
Fig. 6.
0 12 3 4
Volume of phage supernatant (mis)
— FDTSCFVDT3
■° — FDTVH01-3
— FDTPs/Xh
Fig. 7
O.D.
405 nm
1 2i
0
200 400 600 800 1000
Coating concentration ( pg/ml)
FDTSCFVD1 3(lys)
FDTPs/Xh (lys)
FDTSCFV013(BSA)
FDTPs/Xh (BSA)
1200
99
EP0 774 511 A1
Fig. 8.
Cleavage
Site
fd -gene III ¥ fd-CAT2 1
leader ' polylinker
-1 +1 +2
HSAQVQLQELE I KRAAAE TV
--CACAGTGCAcaggtccaactgcaggagcfcgagatcaaacgggcggccGCAGAAACTGTT
fd -gene III
ApaLI
PstI Sad Xhol
Not I
Fig. 9.
O.D. 405 nm
a)HEL b)TEL c)HUL d)BSA e)
Antigen
100
EP0 774 511 A1
Fig. 10.
MKYLLPTAA
GCAnXaCAAATTCTTATITC
10 20 30 40 50 60
AGLLLLAAQPAMAQVQLQES
GCiGGflTrorgmaciaxr^^
70 80 90 100 110 120
GPGLVAPSQSLSITCTVSGF
GGACClG(X!CnGGTGGa
130 140 150 160 170 180
SLTGYGVNWVRQPPGKGLEW
TCATTAACaGGCTATO^
190 200 210 220 230 240
LGMI WGDGNTDYNSAL KS RL
CTQQGAATGATITOG3GriGAT3GAAACACA^
250 260 270 280 290 300
SISKDNSKSQVFLKMNSLHT
A3CA0XaGCAA3GACAAC^
310 320 330 340 350 360
DDTARYYCARERDYRLDYWG
GATCACACAGCCA3GT[AC^^
370 380 390 400 410 420
QGTTVTVS SASTKG PSVF PL
CAA3GCA2CAa3GTCACC^^
430 440 450 460 470 480
APSSKSTSGGTAALGCLVKD
GCACHTIXXTCCA£G£G^
490 500 510 520 530 540
101
EP0 774 511 A1
Fig. 10 con W)
YFPEPVTVSWNSGALTSGVH
550 560 570 580 590 600
TFPAVLQSSGLYSLSSVVTV
ACCITCC033C7IGTCCTA.C?^
610 620 630 640 650 660
PSSSLGTQTYICNVNHKPSN
(XCIOTGCEGCIT^
670 680 690 700 710 720
TKVDKKVEPKSS * *
ACCAAGGTQSACA AGAAAGrrTG^
730 740 750 760 770 780
MKYLL PTAAAGL
AATICEATITCaAQG/^^
790 800 810 820 830 840
LLLAAQPAMADI ELTQSPAS
TCTIATJACIOSCTCCX^
850 860 870 880 890 900
LSASVGETVTITCRASGNIH
COTTICTGCmUIG^
910 920 930 940 950 960
NYLAWYQQKQGKS PQLLVYY
ACAAITATITOGCAIGGIATC^^
970 980 990 1000 1010 1020
102
EP0 774 511 A1
Fig.10conf:f2)
TTTLADGVPS RFSGSGSGTQ
1030 1040 1050 1060 1070 1080
YSLKINSLQPEDFGSYYCQH
AATAITCTCICAAGATC^
1090 1100 1110 1120 1130 1140
FWSTPRTFGGGTKL EI KRTV
1150 1160 1170 1180 1190 1200
AAPSVFIFPPSDEQLKSGTA
TOGCIGCACCATCIGTCIT^
1210 1220 1230 1240 1250 1260
SVVCLLNNFY PREAKVQWKV
CCIUIGITCIGTD3CCT
1270 1280 1290 1300 1310 1320
DNALQSGNSQESVTEQDSKD
1330 1340 1350 1360 1370 1380
STYSLSSTLTLSKADYEKHK
1390 1400 1410 1420 1430 1440
VYACEVTHQGLSS PVTKS FN
1450 1460 1470 1480 1490 1500
R G E S * *
A2a303GM3A3TCM!A?IAAGAATTC
1510 1520
103
EP0 774 511 A1
Fig.10cont(3)
Sphl
Hind3
Sphl
Sac2
Pst 1
PelBl
BstE:
VHD1.3 |
Sail
HuCHl
Sacl
PelB|
Xhol
Vllys | HuCkappa
V- - si '?. .
1 «
II
FabD1.3 in pUC19
Fig. 11.
2-A
3 h
0
Z
Vector
0 FdScFv(OX)
d Fd VHCH1 (D1.3)
0 Fd Fab(D1.3)
□ Fd ScFv (D1.3)
104
EP0 774 511 A1
105
EP0 774 511 A1
Fig. 13.
QVQLQESGGGLVQPGG
CAG GTG CAG CTG CAG GAG TCA GGA GGA GGC TTG GTA CAG CCT GGG GGT
PstI
SLRLSCATSGFTFSNY
TCT CTG AGA CTC TCC TGT GCA ACT TCT GGG TTC ACC TTC AGT AAT TAC
YMGWVRQPPGKALEWL
TAC ATG GGC TGG GTC CGC CAG CCT CCA GGA AAG GCA CTT GAG TGG TTG
GSVRNKVNGYTTEYSA
GGT TCT GTT AGA AAC AAA GTT AAT GGT TAC ACA AC A GAG TAC AGT GCA
SVKGRFTISRDNFQSI
TCT GTG AAG GGG CGG TTC ACC ATC TCC AGA GAT AAT TTC CAA AGC ATC
LYLQINTLRTEDSATY
CTC TAT CTT CAA ATA AAC ACC CTG AGA ACT GAG GAC AGT GCC ACT TAT
YCARGYDY GAWFAYWG
TAC TGT GCA AGA GGC TAT GAT TAC GGG GCC TGG TTT GCT TAC TGG GGC
Q G T L V T V
CAA GGG ACC CT G GTC ACC gtc
BstE I I
ggggsd i E L T
ggcggtggcggatcggac ate GAG CTC ACC
SacI
SLGDQASI
AGT CTT GGA GAT CAA GCC TCC ATC
VHSNGNTY
GTA CAT AGT AAT GGA AAC ACC TAT
GQSPKLLI
GGC CAG TCT CCA AAG CTC CTG ATC
GVPDRFSG
GGG GTC CCA GAC AGG TTC AGT GGC
LKISRVEA
CTC AAG ATC AGC AGA GTG GAG GCT
FQGSHVPY
TTT CAA GGT TCA CAT GTT CCG TAC
s s gggg^ggggs
tCC tea ggtggaggcgglt caggeggagg tggct c t
QTPLSLPV
CAA ACT CCA CTC TCC CTG CCT GTC
SCRSSQSI
TCT TGC AGA TCT AGT CAG AGC ATT
LEWYLQKP
TTA GAA TGG TAC CTG CAG AAA CCA
PstI
YKVSNRFS
TAC AAA GTT TCC AAC CGA TTT TCT
SGSGTDFT
AGT GGA TCG GGG ACA GAT TTC ACA
EDLGVYYC
GAG GAT CTG GGA GTT TAT TAC TGC
TFGGGTKL
ACG TTC GGA GGG GGG ACC AAG CTC
E I K R
GAG ATC AAA CGG
Xhol
106
EP0 774 511 A1
Fig. 74.
AnHgen
Fig. 15.
5 1 END
R T P EMPVL
TCT CAC AGTGCACAA ACT GTT GAA CGG ACA CCA GAA ATG CCT GTT CTG
ApaL1
3'END
K A A L G L K
AAA GCC GCT CTG GGG CTG AAA GCGGCCGCA GAA ACT GTT GAA AGT etc.
Not I
107
EP0 774 511 A1
Fig. 16 ( 1)
Pstl/Xhol
BamHI
A
signal cleavage
site
BamHI
J
BamHI
L
ScFv PCR product
Q V Q L Q E
TTT AAT GA G GAT CCA CAG GTG CAG CTG CAA GAG-
BamHI Pvull
K L E I K R
AAGCTT GAG ATC AAA CG G GAT CCA TIC
Hindlll BamH l
Fig. 16(2)
A
( 1 834) .5' GAG GGT GGT GGC TCT
•( •! <•
m •«
4? tl
C "
C "
"C " ACT 3 4 ( 1839)
B
(2284) 5 ' - GGC GGC GGC TCT
- GGT GGT GGT "
" GGC GGC "
GAG " " GGC "
GGT "
GGC "
GGT "
GGC " 3' (2379)
«» <•
Reverse complement of mutagenic
oligo G3Bamlink
5' GAG GGT GGC GGA TCC
-r I
GAG GGT GGC GG 3
108
EP0 774 511 A1
1) PRIMARY PCR
VH1BACK
VK2BACK
Fig. 17.
inttiiii
cDNA
VH
CH
VK
CK
VH1 FOR
MJK1 (2,4,5) FONX
hQavy
kappa
2) ASSEMBLY PCR
VH1BACK
MJK1(2,4,5)FONX
linker = ( gly • gly • gly • gly-ser )^
♦
3) ADDING RESTRICTION SITES
VHBKAPA10
, i . i :
JK1I2A5)NOT10
♦
Apa L1
NoM
23
109
EP0 774 511 A1
110
EP0 774 511 A1
CI
CD
CO
l
ID
Q
CL
LO
0 s "
Fig. 19.
20
.A
10-
0
fd h-PDGFB R
fdPsBs
No phage
Unlabeled PDGF nM
m
EP0 774 511 A1
Unlabeled PDGF nM
OS-/
Fig. 22.
0-6-
04-
Q
O
02-
00 V
pCAT-3
pCAT-3 ScFv D1-3
fd ScFv D1-3
112
EP0 774 511 A1
d
M
M
113
EP0 774 511 A1
WDocooiaiocoai
cococococncocQcn
8888888
O O O G
X X X X
32
X
>>>>;>£>>;>>>
8888888888888
o o o o n
S X X X x X
O O O O D D
X X X X X X
X
^ a - M Q Q Q Q
OOOCJOOOOOOOOOO
OL QL OL
g< <
u o
<< < «t
a a
t* PS *
< <
u u a
< * S
*: * <
Q Q O
^ ^
U. h* b«
« bd K
a a w
8
>s
4
60
O
C
a>
cr
0)
Q Q O
*: w: u:
Cu Eh U«
^ US
a a a
*»■ c s
CSS
u u p«
E-« Erf M
Q E< f> b. o J
o 5 9 & >? * P
co g co o co oo
c
6
o
o
o
CO
£g
CO CO
< <t a;
poo
a. a. t\
J J J J
M M H «
< < a a
CO
o
33
a a o a a a
a a
o
V
01
O Q
o a
o a
o a
Q Q
a o
Q D
or a»
coo
« *: x.
b k bi
y: stf w
O O Or
o
Lb
a
a d
o o
ooooooooo
X It * X X X
ta iii w w to Da
J J M
O O
a § & a & §
o a a a a c»
x x. w. k *
o
v.
.a
E
o
114
EP0 774 511 A1
(I U O V U
j*: j< jk: *
— < >i
0 o
0 0
g
8
g
4) <U 0) 4) flj 0, <U
j< j< J*
■*-« -»i -H - i
o
~i — i
X X
o o
t O D «i
j< a; j<
-H **H *ri *«-t
•— ( —1 • — * «
S S S g § S g g S S £
X
0
X K X
O 0 o
w > > > H M M
> ft M H > > >
M>>>>>>>
5 > > > > > >
W \ >v "v. >v "-v
>>>>>>>
X
< < *r «< *< <
S 2 S 2 S
H M IH H M *5 M
U H w w u ri w
ds3dd3 3d
gggggge
< < < oo o g o
o o o o o s *
k. U. h. U. U. £ fc
X
33
< A *
^ a! a
W K W W
c5 S
«s <x o <
o o o o
U. Uj Ui Ll.
U W W U [J
S3 d d J
gggg
8 8 8 8
L [l Lu h
5*1
P
6
8
S « W
3 S3 3
gee
< «< <
goo
tL u. u.
2s3g
o 8 3
U, C
f< ^ h
_J J J >- X
a & a o. u
2 >< W H H
5 CD 03 to tO
H H h B
* u * 5 S 5
a o. a £ £ 2
«j «, uj uj «j o to w to K P ° 03 °
88888888S8888888
t* t«
J J J J J
a a a. <x <X
CD (D CO CD en CD
H co oi to co co cd ro
< aj a: co q d
0}t0t0(QID(QtaU>(OCOt0(3tOtO(ZlD)
gggggggggggggggg
CO CD CO TO 0$
888
X X
to co
S 6
co a
88
X
a a a
CO CD CO
b, Ut lu
O O O
a a a
888
888
888
X X
CD i-3
^ O CD CO CO
q- CD CO CO CO
H > H M
Q u to w
VJ O CD 0) CO O)
CO CO 03 CO CD nj 05
^ 4 4 < 4 <
CL fX CO CO CO CO
CO
O)
o
c
a>
a>
CO
o
c
o
o
E
o
Q Q Q Q □ Q Q
a
ft
CD
5
<0
-c
u
o
o
XXX
•J J A
X X >* >* >*
El u It tr »r
C Vi CO CO CO
CO CO CD O) CQ
t> ^ M M »H
CO CO CO CO CO
03 03 03 CD CO
CO CO 0) DO
a. < < a. <
CO CO CO CO CO
XXX
J t-3 tJ
* £ £
2 2 2
CO CO CO
0) 10 CO o)
►HUM
co to co
CO CO CO co
CO CD 03 0)
< «* < < . , _
CO CO CO tD CO CQ
2 X
3 j
X
-3
CO
3
X X X
(0 CO CD
to 03 03 to «n
O) 03 O) to
CO
, Ai ^ to to CO
tO to CO CO CD CO CO
2 2 2 2
m m 2 S S 2
w w w M a
o o o 5 F
a a i s
to to co CO
H
u W CO Ed CO CO
O O O O 0
a. a a a fi.
co CO CO CD CO 01
S to co to to
2 z z a x
g
. *t < *t
a a a a
CD CO CO CO CO CO 0)
aaaoapp
QQQQQQPQQQ
115
EP0 774 511 A1
Fig. 25.
HEAVY CHAIN
A
B
C
D
E
F
G
H
a
b
» J * • • ■ • '» *"» • *"_
•:■•-*■ ■ •; ■••:„■■
> « _ • * 9 * * * * ■
* ^ • * • • • • • * •* *
■ • • ■ • * * • • *
. .-.\- 7, v. f.*-,-"
•\ ..*•• ?***•.
■ # • ■ * * ■ ■ ^ »• - «
■ • • ■ * ^ * \*'
c
d
151
i
G
f
g
I
mi
» > ■ ♦ ♦
OD 405nm inEL,SA
0.2-0.9
0.9-2.0
>2.0
116
EP0 774 511 A1
Fig. 26(a)
Hindlll
PlacZ
pHEM
4523 bp
co/Elori
AMBER
EcoRI
Fig. 26(b)
PelB leader 1 -pHEN1 polylinker -
-1 +i
LLAAQPAMAQVQLQVDLEI KR
--TTACTCGCGGCCCAGccggccatggcccaggcgcagctigcaggccgacctcgagaccaaacGg
Sfil Ncol Pstl Sail Khol
1 c-myc tag 1 | — fd-gene
AAAEQKL I S EEDLN G A A (E) T V E
gcggccGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAA-
amber
Not I
117
EP0 774 511 A1
Fig. 27.
ApaLI Not I
P L
/
\
VH
CHI
rbs
— o —
VK
CK
\
\
pelB
\
Fab
\
\
VH
CHI
' heavy chain
! I
VK
CK
^ liqhf chain /
\ /
\ /
\ /
Sfi I Nofl
. . Amber
lacZ pelB tag
gill
118
EP0 774 511 A1
Fig. 28.
119
EP0 774 511 A1
Fig. 29.
Anti- Anti- AnH-
c-MYC CK Fab
i 1 i — ; 1 r— 1
+ - + - + —
i - ... i ■ — i
120
EP0 774 511 A1
Fig. 30.
-1 > — i > 1 ■ 1 — 1 1 ■ 1 ■ 1 1 — i
-4-3-2-10 1 2 3
[tog] lysozyme dilution ( 0 = 1ug/ml )
Fig. 31.
1 ' 1 1 1 ' 1
0 12 3
[log] Lysozyme dilution (0=1ug/ml)
121
EP0 774 511 A1
e
LO
QJ
C
o
00
3H
1
38!
Phage 013.21
Phage o13.28
Phage o 13.30
^ Phage 013.36
I I Vector control
0
Fig. 33.
QJ
d
CO
O
00
10
e
t£ 0-8 H
0-6-
0-4-
02-
Phage o14.6
Phage o14.20
H Phage 014.21
H Phage 014.22
l~l Vector control
00
122
EP0 774 511 A1
Fig. 3U.
123
EP0 774 511 A1
Fig. 35.
B
a b c
a b c d e f
200
925
69
46
200
925
69
46
Fig. 36.
M > > -L + L M V K V H -L + L M
: ;.l
3fe*<w>>.' 1
Fab
v —
scFv
124
EP0 774 511 A1
x x
X X x
nuso^ ao
125
EP0 774 511 A1
Fig. 38.
126
EP0 774 511 A1
Fig. 39.
Coating Concentration (ug/ml)of Lysozyme
Fig. 40.
en
ro
CL
m
>
LJ
to
0 s
20%-
15%-
10%-
5% J
0%
Starting %
Elution %
4.8% 4.3%
30 ug/ml 3 ug/ml 0.3 ug/ml
Coating Concentration of Lysozyme
127
EP0 774 511 A1
Fig. 42
A
12 3 4
B
12 3 4
C
1 2 3
° N
1 2 3
< *-
• it
||
1 v
)
128
EP0 774 511 A1
Fig. 43.
SN
□ SN + sCD4
DID SN+gp120
fd-tet fd-CD4-V1 W-CDW1V2
129
EP0 774 511 A1
o
H < fO
o
F 1
H
<
c
o
r F> t *
O O w
cn
U O H
<
H
CO
i
o o o
O O <
, — j
<
F 4
<: H c/j
o u w
H
o
F > F f> 0—*
OOP
<£-«>«
O
O
f 1
tH ft« cy
ft, H »-3
F \
o
O
D-i
O O rH
O O to
r .
tit
/ n r ^ r"
U U u
f fx r \
O O >t
<.
CO
s n h
H < O
o
o
<.
KJ) \D \D
r \ r r\ i i
CJ O M
r .
tH
A.
cy
o
O O CD
o
tH
Ai
f — N
H <I CO
t
<Z
r .
tr 1
1 1
1 — J
\£>
C_i
A. tH
OOP
rH
o
o
CN
O O W
H <C CD
O
O
j— i
r_i
fcH >1
* \ f rx ^
U U M
<.
w
tH ^ tH
O O co
<.
< E- 1 >»
o
O
r— I
O O ^H
^< f- *x
< H i-3
O
o
r r\
o o <c
O O rH
<
A 4
o
< H M
F 1 <T ■
£h < CO
<
1
1 — \
F \ F F* F^
O O
o
O CJ >
O
<
F*
CH
HH
O
< H H
r~
o
O
cx
t o
U J
F \ F fX ( ■
O O H
O O 0
rH
o
o
CM
f fX F \ /* 1
o o CU
H < 00
tH
<
Eh
<i tH CO
r- — -
H < CO
o
o
P
O O M
O O rH
<
f-H
fX F fX F1 V
o o CU
O O <
F FX
o
o
r ri
o
tH CO
CJ? O >i
F— 1
P
o o o
O U H
H
<
cu
O O H
o o o
o
F FS
o
i-H
O O D-t
o
H < O
o
o
F F^
o
O
-rf* f M F/\
< H W
r ^ r f-n »
u o ^
o
o
« — 1
»
<. fcH >-
O O 0u
rH
O
O
o
CNJ
<- H H
o o co
<
f .
r— (
■»
CJ*
O O tlj
< H >*
r fv
o
F %
o
f ,
<- tH CO
< M J
o
o
t fX F \ --Ft
o o <,
O O — <
<
E-i
>1
< H rH
H < (0
o
O
» — !
tn <C CO
O O >
o
o
o
F PX F \ "N*.
o o >
H < P
<
o
o
Eh < CD
O
o
o
JH
o
FX F fX F - •
O O XI
UJ
U O ^
■ I \
o
o
\ j
CNJ
< H Eh
OOP
— t
o
o
CT»
OOP
< H «H
o
o
M
tH <v< UJ
o o o
<
O O rH
H < cO
o
O
c
< H H
O O -H
<
Eh
*
» — i
F \ F F\ *
O O X.
O O <
o
O
F F%
o
F» - F*
< Eh Eh
o o >»
o
o
CO
O O CO
O O rH
<
Eh
fx f rx i
O O iH
O
* r% * V F FX
o o o
o
<
H
O
O O <N
£h < Jh
* i
o
O
rH
F FN F \ f A
o O CO
■> V p Fx 41 t
O O <U
rH
CNJ
< h >i
H < CO
o
O
< r h1
o o c
o
o
O O w
ft, t -1 "H
o
o
p V p PX P r-\
o o o
H
<
tH
s, n Uj
o o c
o
o
CO
r fx f \ # a
O O co
< H *-i
<
Eh
-H
<. CH >t
o o o
o
O
N P H
o
OOP
/ — ^
o
O
' >
r 'i r n /ii
(V)
oo
H
<
/II
rH
tH ri-.
CH
O \D \— \
rH
<
«^r»
2l.
o o c
o
O
til
o o
< 4
M
tH w*l
O O O
H
<
tH
c_< J
n h
o o »— I
O
O
C 1
M
fori i
<
H
f>1
£H ^-4
OOP"
<
tH
t n r i r fi
O LJ
o o c
O
O
i *
W
r i v-r* r*
o
^ H rH
O
O
CU
r . ^«
R, n UJ
t N
O CD
— J
1 1
<
H
r r\
\JJ
o
^r 3 c_i f-r 1
k n ^
"3»r rzT
rH
o
O
f i
CM
r \ rh t .
U U H
U U 1 — 1
o
O
«— ■
#c
*V C-l
(Q, tH
o o
<c
Eh
r .
r s n
o
O
CU
V»J UJ
Eh
<
"< H CO
H
<
< H ^
O O W
O
o
Jh
H < U
< H -H
o
o
o o x:
o
o o ac
O
<
Eh
O
< Eh H
rH
H < ^
O
o
O
Jh
CT>
H < >
O O <D
rH
Eh
>i
rH
O O »H
H < CO
Eh
<
Eh
o o o
E-i < M
O
O
>.
H < >n
O
O
4 \
O O rH
H < Eh
O
O
o
O O O
O O CJ
H <
u
Eh < M
H < JG
o
o
0)
o o <u
h < Oj
Eh
<
CO
< H CO
o
m
O
o O
O
o
o
<
<
o
o
o
o
o
o
H
TO
<
Eh
O
<
o
Eh
tH
H
O
<
O
<
H
O O
'Zl O
O
<
H
O
O
o
<
Eh
O
o
o
<
Fh
o
CM
CO
o
o
o
en
CM
o
CO
CM
Eh
o
Eh
Eh
<
Eh
Eh
<
O
tH
O
O
o
o
tH
o
H
o
<
o
o
<
o
Eh
O
<
o
<
o
O
o
o
<
t n
u
r-v. tH
rr>
cc
C — i
tH
f" j _<*F* y« ■
fcn <. CD
r o
r— 1
LO
r-s
c__.
tH
■
' — !
CO
CH fZ. •—.
tH
r \
c_->
r n
CJ
r n
CJ
CO
<£. fcH rH
r -s
>
1
CH
tH
/ \
■ i
r n r \ «
OOP
rH
rll
C— i ft \
O
tH ^-s
rn
yjj
f n
*o
CJ
U U M
FH
C
H
Ui
CJ CJ >H
C__i
rH
itf 1 FH
f-U tH
r-J
CJ
I 1
r-»
r ^ r n /i\
CJ CJ CD
O
FH
tH
FH
tH
r . — r* r
fcH «, CQ
C 0
>
!
f r> r )
T \
f 0
r*
u
CJ CJ r>1
r )
rH
Vj f
<f FH
rH
FH
I — \
^ J
V-J vJ r— |
o
V — ' \-J
CO
m
r-s
tH
CM
f ri r \ rn
CJ CJ CJ
a
r-i, tH
r >
r r»
to
^r" CH t i
r-v, tH M
v — '
r ^
J
CJ CJ UJ
fcH
r^. t -1
CH
fcH
f *">
W
CJ
A.
t 1 K U J
f 1
\JJ
tH
r^*
CJ CJ QJ
r_i
n
>
>
CH
n s £
fcH
r pi r \
O
F
tH
^«r*
M.
fcH
cH <, CU
o
a
tH kL,
r— 1
O
O
C
k, r (J>
tH
CO
tH
IT 4
tH
CO
/ n r \ t ■
O O w
o
<
rn r
c_u
/ — V
^ J
fcH
A,
V — J
**** r_i ^-Y*
N P <,
r~vi
CD
CO
tn ^i.
Hj
CM
r i
fcH
rH
r \ r r\
O O CO
A.
JG
T
r— 1
LT)
r r.
o
CJ
CD
r \ r r\ j
O O rH
CU
rn n
tH
f rj r \ — <•
U U K.
o o
C
c__,
tH
A,
JH
tH O
tH
>
I
< fcH
rH
CJ
r v rn < ,
O O H
o o
tn
tH 1
CJ CJ (Xi
o o
A,
j
^_ T
t .
>-i
CH ^i, »-H
tH
1
o o
CD
r ^
O
CJ
fcn ^ ro
fcH
Eh <
CO
A.
r,
p 1
•a
tH
CJ O *>
r— ^
< — >
< fcH
/ — \
v J
tH
r— (
v >
r__i ^*
tH Pi
j» i
CD
CM
o o
rH
rH
tH
CO
% «r
/ ri r i i
CJ CJ rH
tH
CO
o o
o
ID
r n
cj
CJ
CJ CJ CJ
r-si
u
o o
>
r-v,
k, n U
r ^
CD
o o
rH
r \
CJ
CJ
i
r— 1
r ^ t n t ■
CJ CJ H
CH
tH
CO
o o
o
O
/■ i
O
O O CU
>
1
fcH <
>
O
O
P t0
O
rH
O O -1
r n
O
CJ
rH
O O r— 1
O
o
o o
o
O
r \
O
r r\
O
o o <.
f rj
o o
>
tH
>H
-*-r* C j r^TX
P CP
fcH
I
' — '
o o
rH
o
O
^— •
r**%
O O H
Eh
rH
o o o
o
rQ,
tH
tH
o o <.
t n
O
a
Eh <
M
in
tH
M
in
o o c
tH 1
to
o o
CD
CJ
CJ
<JJ
^vi tH U}
r \
<
H <
CO
r^
tH 1
r r\
UJ
F^, tH ft.
f r»
O
u
O O
>i
r ■
fcH
t ,
M
O O C
fcH
I
o o
rH
o
r r<
O
CD
<z fcn co
<«
Eh
o o
O
<.
F FX
co
<i fcH <,
r ,
fcH
CP
Eh <
>
F \
o
o
CJ»
P \ P FX r
O O in
u
tH
O O
rH
CJ
r \
CJ
M
r \ r n
O O JZ
fcH
<
O O
o
/-~\
CJ
f n
CJ
v )
r . r .
r^, tH CH
fcH
to
o
< H
>
tH
A.
CO
fcH ft, >1
f r»
V_/
rH
O O
rH
o
o
>1
m
O O r— I
<
O O
o
p>
CH
<
o
/ n r \ rn
O O O
<
to
O O
>
IH
<
>H
Z—i
tH ft, P-i
o
O O rH
O
o
x:
CJ CJ r— l
o
O O
o
<
Eh
Eh
rn r i r n
CJ CJ CJ
u
< H
O
o
P
#^r* c ■■ ft \
ft. tH CD
Eh
>1
O O
CD
H
<
CD
fcH ft. rH
<
H
Eh <
CO
O
o
rH
^ U h
<
Jh
o
fcH <
>l
Xm F 7
<
Eh
}H
C-J
•CT rH r3
Eh
>i
O O rH
CO
o
o
x;
r— i ft -
C 1 r««i Vi/
/<
tH
CO
O O
o
<
H
m
ri rn j
O
rH
o o
>>
o
O
rH
r_i ^ s,
fH r-i, ^-n
<
CO
o o
rH
fcH
<
CO
V_J r— l
o
>
o o
O
o o >
ci t j f n
V_> vJ <o>
o
CO
< H
>
<
Eh
C ' r< r-»
O rH
O O rH
o
O
x;
^J i
o <
o o
o
<
H
f— i CH
ft, tH tH
<
U
tn <:
>
<
Eh
P
r ^ rn /<)
CJ CJ UJ
o
CD
X . F
O O rH
o
<
tH rH
fX
V /
tH ft, JH
<
CO
CO
CO
o o
CJ2
o
O
o
CO
tH fti FH
o
CU
a: P
u
tH
<
>l
in
ft, CH ,J
CO
o o
CD
o
O
rH
CH trf* rll
o
<
CO
o
o
O
fH *^
CH r-^ » — <
o
p
o o
i-i
H
<
o
CH <5* rn
r— • r-*„ UJ
fcH
rH
o o
CD
O
o
<1 Fh -h
o
O
Eh <
CO
O
o
Cu
<
M
O O rH
<
Fh
Cj
Eh <
o
CD
H <
CO
o
O
CD
< Eh CO
CO
o
O O >
o
fcH
<
CO
o
O O <
H
M
r-
CO
O O
<
IH
u
m
in
< H O
O
x:
o o x:
o
O
x:
O O M
fcH
< fcH
fcH
<
Fh
fcH
O O Cu
O
P
o o
rH
o
O
M
ft Eh (0
<
CD
H <
CO
o
O
x:
< H >i
O
hH*
o o >
<
H
IH
< P
o
M
o o
u
o
O
P
< H D
o
CD
o o
x;
tH
<
CD
< Eh rH
tH
to
< iH
o
O
rJ
o o o
130
EP0 774 511 A1
o
CJ
a
■ — (
CM
H
<
CO
CJ
cj
>
CJ
cj
a
CJ
H
<
<
co
H
CJ
CJ
re
CJ
CJ
c
o
<
H
CO
<
H
<
r-
CJ
CJ
o|
[fil
H
o
CJ
a
H
<
cj
u
o
H
<
H
o
< H
r*
H
CU
O
CJ
H <
03
CJ
CJ
» — 1
CO O <
H
CO
CJ
>l
H
<
CJ
o
CJ
CJ
<u
H
x:
KD
H
<
a.
H
<
u
<
>i
<
<D
<
— I
H
HH
<
H
fC
O
CJ
CJ
< — 1
CO
CJ
CJ
<
V£>
cj
o
a
<
H
• — j
cj
U
CJ
<c
a
<
CO
cj
CJ
<
o
p
<
H
1— 1
O
cj
U
CJ
r-
Eh
<
>H
VD
O
CJ
<
H
CJ
O
c
<
H
CJ
CJ
CJ
<
H
cj
o
<
o
cj
CJ
>!
o U H
KD
CJ
CJ
CJ
E-»
>H
CJ
CJ
x:
<
H
Eh
a cj
H
M
CJ o
O
o cj x:
m
E-i
o cj
P
H <
<D
O O
h3
O
CJ
CO
CJ
CJ
•— I
O
o
H
<
to
CJ
CJ
rH
o
CJ
CJ
o
u
co
H
>i
<
£ i
CJ
a,
<
CO
CJ
CJ
<
H
CJ
CJ
i— <
CJ
CJ
CJ
o
r-
o
r*
o
m
r-
O
CJ
CJ
u
CJ
CJ
o
CJ
CJ
CJ
o
<
o
<
CJ
<
CJ
CJ
H
u
u
-B
u
<
CJ
H
o
CJ
u
CJ
CJ
CJ
u
CJ
CJ
CJ
CJ
CJ
Eh
CJ
<
Eh
CJ
Eh r-H
CJ CJ
CJ P
CO
<
CO
<
CP
<
CO
>1
<D
r-l
f-H
<
CJ
CJ
<
CJ
<
CJ
CJ
<
CJ
H
CD
•-^
«— J
CO
>
U
H
<D
CO
<C
CJ
o
CJ
m
<
CJ
CJ
O
H
H
CJ M
cj
H >.
O • — !
CJ CJ
H >.
O «H
CJi^
< >
CJ ■ <
CJ CD
< JC
< On
131
EP0 774 511 A1
Fig. 45.
r ApaL1
T
rbs
VHD1-3
333 bp
Gene III leader
BstE2 rSad
SphI - SstI
rbs
Seal
rXhoI
Not I
VkD1-3
310bp
Pel B leader
N- Terminus of Gene III
Fig. U6.
2 000
E
c
CO
Q
O
1-475
0-9500
-sc Fv DT3
-Fv D1 3
-Fv D1-3 (AS -Stuff er)
0-4250
-0-1000
Dilution factor
132
EP0 774 511 A1
Fig. 4 7
mRNA
mRNA
HuIgG1-/»CH1F0R
HuCIFOR
1st strand cDNA synthesis
t
1st Strand VH-CH1 cDNA
t
1st Strand VL CL cDNA
HuVHBACKMix
HuVLBACKMix
HulgGI 4CH1 FOR
HuCIFOR
HuVHBACKMix
Primary PCRs
t
VH
H 1
C
T^ gpelB Leader " v ,
c
CL
^ 1
HuCIFOR
Linker DIM A
f
HuVHBACKSfiMix
^ _VH
I
Temperature cycling followed by
amplification
♦
CH1
pelB Leader
I
VL
CL
I
I
1
Reamplification with primers
containing restriction sites
HuCHFORNof
t
Assembled Human Fab with 5 1 and 3' restriction sites
133
EP0 774 511 A1
pelB VHD1-3 Hu CH1 myc pelB VKD1-3 HuCK
C. Sequence of linker region
3' Human CHI and hinge
KPSNTKVDKKVEPKSSTKTHT
MCCCCAGCAACACCAAGGTC^
myc peptide tag
SGGEQKLISEEDLN**
TCAGGiAGGTGAACAGAAC^^
pelB leader
MKYLLPTAAAGL
AATTCTATTTC^AGGAGAC^
+\ \+ 5'Vk
LLPAAQPAMADIELTQSP
TATTACCTGCTGCCC^CCAGCGATGGOCGAC^
134
EP0 774 511 A1
Fig. 49.
mRNA
mRNA
HuJHFORMix
HuJIFORMix
t
1st* strand cDNA synthesis
t
1st Strand VH cONA
HuVHBACK2Mix
1st Strand VL cDNA
HuVLBACK2Mix
HuJHFORMix
HuJIFORMix
HuVHBACK2Mix
t
Primary PCRs
t
- i — i
VH
"'scFv Linker
i h
■9 dzn
VL
■ i 1
Linker DNA
t
HuVHBACKSfiMix
VH
Temperature cycling followed by
amplification
HuJIFORMix
t
scFv Linker
VL
C
I
T
Reamplif ication with primers
containing restriction sites
HuJIFORNotMix
♦
Assembled Human scFv with s'and 3 1 restriction
135
EP0 774 511 A1
Fig. 50.
CLONE B1
Concentration
Concentration
136
EP0 774 511 A1
Fig. 51
relative
OD405
111 Mil m ■ M •* ■ MLJ M i ■' H i ■ -I t ■. I i M ,
123456789 10
Phage clone
F/g. 53.
H Sp PstI BstEII H SP
m&ssksssssssksaA
BstXI
i
Xhol
L
B E
V
Pel B VH
PelB VL
Myc Tag
137
EP0 774 511 A1
a
Q
<
E-<
E-«
E*
>h
>
J
a
ft
co
o
a
o
a
<
W
H
Z
o
OQ
<<
U
E-"
H
Eh
>
E-
fa
O
>
CO
<
CO
CO
<
ft
CO
a
Eh
a
H
Q
Q
CO
CO
CO
Eh
<
>H
H
H
Eh
O
Q
ft
W
a
a
CO
CO
o
Q
<
a
Eh
►J
CO
>
w
a
CO
<
CO
CO
CO
ft
CO
a
Eh
w
H
Q
fa
00
z
CO
Eh
O
>H
ft
ft
CO
Eh
w
CO
a
a
>H
W
CO
CO
CO
H
CO
CO
CO
>
CO
CJ
Eh
H
Eh
>
w
o
ft
CO
2
<
ft
CO
o
Eh
w
Q
a:
M
w
Eh
CD
O
O
fa
Eh
Pi
ft
Eh
CO
fa
a
o
a
>H
>■
CO
o
fa
Q
fa
ft
a
•J
CO
s
H
CO
>H
o
Eh
O
CO
a
CO
o
co
fa
oc
CO
ft
>
O
W
Eh
O
u
o
fa
Eh
ft
CO
CO
a
u
>H
>H
Q
>
fa
Q
W
CO
U
CO
CO
H
J
CO
Q
CO
o
CO
Pi
co
O
CO
>
w
O
o
E-
CO
CO
o
a
o
>*
>*
Q
w
<c
w
s
CO
co
H
CO
CO
Eh
O
CO
O
CO
O
CO
(x,
>
a,
>
CN
2
138
EP0 774 511 A1
European Patent
Office
EUROPEAN SEARCH REPORT
Application Number
EP 96 11 251G
DOCUMENTS CONSIDERED TO BE RELEVANT
Category
Citation of document with indication, where appropriate,
of relevant passages
Relevant
to claim
CLASSIFICATION OF THE
APPLICATION (Int.Cl.6)
SCIENCE,
vol. 246 , no. 4935, 8 December 1989,
pages 1275-1281, XP000083689
HUSE W D ET AL: "GENERATION OF A LARGE
COMBINATORIAL LIBRARY OF THE
IMMUNOGLOBULIN REPERTOIRE IN PHAGE LAMBDA"
* the whole document *
WO 90 14443 A (HUSE WILLIAM D) 29 November
1990
* the whole document, especially example 5
J BIOL CHEM, MAR 25 1988, 263 (9)
P4318-22, UNITED STATES, XP002023948
DE LA CRUZ VF ET AL: " Inmunogeni ci ty and
epitope mapping of foreign sequences via
genetically engineered filamentous phage."
* the whole document *
MOLECULAR IMMNULOGY,
vol. 23, no. 7, 1986, EXETRER, GREAT
BRITAIN,
pages 709-715, XP000616967
GEYSEN, MARIO A. ET AL.; : "A priori
delineation of a peptide which mimics a
discontinuous antigenic determinant"
* page 710, column 2, line 38 - page 711,
column 1, line 15 *
GENE,
vol. 73, 1988, AMSTERDAM NL,
pages 305-318, XP002024990
PARMLEY, STEPHEN F. ET AL.:
"Antibody-selectable filamentous fd phage
vectors : affinity purification of target
genes"
* the whole document *
-/--
1-45
C12N15/10
C12N15/62
C12N15/73
C07K16/00
C12N1/21
//(C12N1/21,
C12R1:19)
1-45
1-45
1-45
TECHNICAL FIELDS
SEARCHED (lntXl.b)
C12N
1-45
The present search report has been drawn up for all claims
©
3
a.
s
2
OS
C
u.
O
ft,
u
Place of search
THE HAGUE
Date of completion of the search
11 February 1997
Examiner
Nauche, S
CATEGORY OF CITED DOCUMENTS
X : particularly relevant if taken alone
Y : particularly relevant if combined with another
document of the same category
A : technological background
O : non-written disclosure
P : intermediate document
T : theory or principle underlying the invention
E : earlier patent document, but published on, or
after the filing date
D : document cited in the application
L : document cited for other reasons
& : member of the same patent family, corresponding
document
139
EP0 774 511 A1
European Patent
Office
EUROPEAN SEARCH REPORT
Application Number
EP 96 11 2510
DOCUMENTS CONSIDERED TO BE RELEVANT
Category
Citation of document with indication, where appropriate,
of relevant passages
Relevant
to claim
CLASSIFICATION OF THE
APPLICATION (Int.CL6)
P.X
METHODS IN ENZYM0L0GY,
vol. 178, - 1989
pages 497-515, XPO02024991
ANDREAS PLUCKTHUN ET AL. : "[34]
Expression of functional antibody Fv and
Fab fragments in Esherichia Col i "
* the whole document *
WO 88 09344 A (CREATIVE BIOM0LECULES INC)
1 December 1988
* the whole document *
NATURE,
vol. 348, 6 December 1990, LONDON GB,
pages 552-554, XP002024992
MCCAFFERTY, J. ET AL. ; : "Phage
antibodies : filamentous phage displaying
antibody variable domains"
* the whole document *
1-45
1-45
1-45
TECHNICAL FIELDS
SEARCHED (Int. CI. 6)
The present search report has been drawn up for all claims
3
3
3
1
c
it.
c
a.
Place of search
THE HAGUE
Date of completion of the search
11 February 1997
Examiner
Nauche, S
CATEGORY OF CITED DOCUMENTS
X : particularly relevant if taken alone
Y : particularly relevant if combined with another
document of the same category
A : technological background
O : non- written disclosure
P : intermediate document
T : theory or principle underlying the invention
E : earlier patent document, but published on, or
after the filing date
D : document cited in the application
L : document cited for other reasons
& : member of the same patent family, corresponding
document
140
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11